Index:
ABSTRACT page 3
INTRODUCTION 5
Breast cancer susceptibility and Hereditary Breast Cancer 5
Genes involved in breast cancer predisposition 10
BRCA1 gene 17
DNA Damage Response and Repair Mechanism 24
BRCA1 and Homologous Recombination Mechanism 33
Pathogenic variants and VUS (Variants of Unknown clinical Significance) 40
Gene expression profiles and different phenotypes of breast cancer 52
Genes MRE11A, OBFC2B (hSSB1), EEF1E1(p18) 59
AIM OF WORK 67
MATERIALS AND METHODS 68
Microarray data analysis 68
1) Bioinformatic analysis 68
1.a- Sigle gene analysis 68
1.b- Pathway analysis 70
2) Validation of microarray experiment 72
2.a) Real Time RT-PCR 72
2.b) Western Blotting 76
Homologous Recombination in vitro assay 77
1) Isolation of hprtDR-GFP HeLa clones 77
2) siRNA and pCBASce plasmid transfection and cell analysis by fluorescent microscopy and FACS 78
3) Evaluation of protein levels 80
4) Cell survival assay and HR assay after ionizing radiation exposure 80
Mutational analysis 80
Selection of the patients 81
DNA extraction ; Design of Primers and DNA amplification; 82
PCR Purification PCR Marking ; Marking Purification 85
Sequencing reaction 86
RESULTS 88
Microarray data 88
1.a- Pathway analysis 89
1.b- Gene analysis 94
2) Microarray data validation 97
2.a-Validation by RT-qPCR 97
2.b-Validation by Western Blotting(WB) 98
Identification of new BRCA1-partner genes in DNA repair mechanism 99
1) Knockdown of BRCA1-parteners genes by siRNA transfection in HeLa hprtDR-GFP 99
1.a-The depletion of OBFC2B, EEF1E1 and MRE11A genes reduces the HR frequency. Analysis by fluorescent microscopy. 101
1.b- Confirmation of the effect of OBFC2B, EEF1E1 and MRE11A in the HR mechanism by FACS analysis 104
2) HR assay and cell survival assay after ionizing radiation exposure 107
Mutational analysis of the genes OBFC2B and EEF1E1 111
DISCUSSION 116
Abstract
M1775R and A1789T are two missense variants located within the BRCT domain of BRCA1 gene, both isolated from familial breast cancers patients. The M1775R variant has widely been described as deleterious by both in silico analysis and functional assays, while the A1789T variant has been identified and classified as probably deleterious for the first time by our group. To investigate the molecular mechanism that may underlie a pathogenetic role for these two mutations, we compared the expression profile, obtained by microarray, of HeLa G1 cells transfected with the two variants. Compared to BRCA1 wt, the M1775R and the A1789T variants showed 201 and 313 differentially expressed genes respectively. Pathway analysis showed that many pathways are modulated by both mutations and that many of them are involved in cancer onset and progression. This evidence indicates that the M1775R and A1789T variants may predispose to cancer through similar mechanisms and this is consistent with the co-localization of the two mutations in the BRCT domain of BRCA1.
The expression levels of nine genes (CDKN1A, EDN1, GPR56, NFkB1, PML, SOD2, MRE11A, EEFE1E, OBFC2B), selected because of their involvement in neoplastic transformation, were validated by RT-qPCR. Moreover the expression level of five proteins was confirmed by Western Blotting (WB).
Our hypothesis is that some genes, coming out from microarray experiment ,are probably implicated in BRCA1 pathway and could be considered as candidate genes of susceptibility involved in familial breast cancer.
We chose three out of nine validated genes (MRE11A, EEF1E1, OBFC2B) for a further validation and test their involvement in DNA double strand breaks repair and in particular in the “Homologous Recombination” (HR) process. The knockdown of OBFC2B, MRE11A and EEF1E1 reduces the HR rate respectively of 28,6%, 41,8 % and 42,3% compared to the controls. Our results together with the knowledge obtained from literature suggest that those three genes might be involved in
BRCA1-pathway.So we decided to do a mutational analysis on two of these genes in individuals belonging to Breast/Ovarian cancer families tested negative for BRCA1 and BRCA2 germline mutation. For the gene EEF1E1 we found three variants known in literature and one never described previously, but none of them are reported as pathogenic.
Introduction
- Breast cancer susceptibility and Hereditary Breast Cancer
Breast cancer is the commonest cause of cancer death in women worldwide. Rates vary about five-fold around the world, but they are increasing in regions that until recently had low rates of the disease.
Although nongenetic factors also clearly play a role in the familial clustering of breast cancer, 5%–10% of all breast cancers can be explained by the inheritance of mutations in one of the two major breast cancer susceptibility genes BRCA1 and BRCA2.[ 1 ] However, a great deal remains to be understood regarding the number of heritable genetic factors involved in breast cancer, the function of such genes, and the prevalence of breast cancer-associated mutations. It is well understood that one of the most important risk factors for developing breast cancer is a family history of the disease but, many other nongenetic risk factors can contribute to disease etiology. They can be categorized as hormonal and nonhormonal risk factors. It has been shown that estrogen exposure is directly associated with risk for developing breast cancer and that a prolonged or increased exposure to estrogen is associated with an increased risk for developing breast cancer. Therefore, factors that increase the number of menstrual cycles are associated with an increased likelihood for developing breast cancer, such as early age at menarche and late onset of menopause [ 2; 3 ]. Similarly, it appears that decreasing the total number of ovulatory cycles can be protective, which can be achieved by moderate levels of exercise [ 4 ] and a longer lactation period [ 5 ]. There are discrepancies in the literature concerning the use of postmenopausal hormone replacement therapy as a risk factor for developing breast cancer. Overall, the incremental risk is small, but there is some evidence that suggests risk increases with long-term use [6; 7]. The age at first live birth can influence the risk for breast cancer development [8 ], in fact there is a greater protection for early first birth and for a larger number of births It has been hypothesized that these data
suggest a protective effect of the terminal differentiation of breast epithelium associated with a full-term pregnancy and that this effect may be most protective at an early age. Finally, there is an association between obesity and an increased breast cancer risk [9; 10 ].
A number of non-hormonal risk factors are associated with the development of breast cancer; however, some of these risk factors may be indirectly related to the modulation of estrogen exposure. One important risk factor is the exposure to ionizing radiation. Young women who received radiation for Hodgkin’s lymphoma have a markedly increased risk for developing breast cancer, with an incidence ratio of 75.3 [11] as compared with age-matched control subjects. In addition, survivors of the atomic bomb blasts in Japan during the World War II have a very high incidence of breast cancer [12], likely because of somatic mutations introduced directly by radiation exposure. In both cases, it is postulated that exposure during adolescence, a period of active breast development, enhances the effect of radiation exposure [13]. A number of studies have suggested that another factor that increases breast cancer risk is the alcohol consumption. Nagata et al. [14] provided evidence that alcohol consumption can increase serum levels of estradiol, suggesting that alcohol consumption is indirectly associated with the development of breast cancer by increasing the exposure to estrogen.
Finally, other nonhormonal risk factors are some dietary factors include high dietary fat [ 15] and “well-done” meat [ 16], but the evidence for these factors increasing the risk for developing breast cancer is controversial because of study bias, discrepant data, and the inherent difficulties associated with collecting neonate dietary-exposure histories. Nevertheless, dietary fat intake may be a factor in indirectly increasing serum estrogen levels, and well-done meat may contain specific genotoxins. Interesting is the hypothesis that nonhormonal risk factors may contribute to breast cancer development in relationship to common variant alleles of a variety of genes. In general the manifestation of multifactorial diseases, such as breast cancer, almost certainly depends on the interplay between environmental and genetic factors. But the
most important factors correlated to breast cancer risk are the family history and mutations in the BRCA1 and BRCA2 genes.
In 1866, Paul Broca was the first to describe a family with a high prevalence of carcinoma of the breast. His wife suffered from early onset of breast cancer and when he made a pedigree of her family, four generations with breast cancer could be identified [17]. The “Broca” report is the first of many that pointed out that breast cancer can be inherited, passing through from one generation to the other. [18]. With the knowledge of today, only in about 5% of all the breast cancer cases, the disease will occur as part of a hereditary cancer susceptibility syndrome, caused by mutations in high penetrance susceptibility genes. Germline mutations affecting one allele of either BRCA1 or BRCA2 confer susceptibility to breast and ovarian cancer with high
penetrance, engendering a Hereditary Breast and Ovarian Cancer Syndrome
(HBOCS). The cumulative risk in the BRCA1-mutation carriers to develop breast cancer by the age of 70 years is 50-70 % , and 30- 40% to develop ovarian cancer. For BRCA2-mutation carriers the corresponding figures approach 40-50% for breast cancer and 10-15% for ovarian. Whereas cancer risk in BRCA1 mutation manifests largely in the female breast or ovary, individuals who carry BRCA2 mutations also have an increased risk of other cancers including those of the male breast, pancreas and prostate.[ 1] Germline heterozygosity for BRCA gene mutation is enough for cancer predisposition. The cancers that arise in mutation carriers appear to lose the second BRCA allele through somatic alteration that must occur during tumor progressor. So a substantial proportion of hereditary breast cancers, about 16% [19], can be attributed to germline mutations in either of the BRCA (breast cancer 1 and 2) genes . Nevertheless, various other genes conferring an increased risk of breast cancer have been identified, including TP53, PTEN, STK11/LKB1, CDH1 with high
penetrance, and ATM, CHEK2, , NBS1, RAD50, BRIP1 and PALB2 with an intermediate penetrance. Some of these genes are involved in multiple cancer
syndromes like Li-Fraumeni (TP53), Peutz-Jeghers (STK11/LKB1) and Cowden syndrome (PTEN) [ 17;19; 20; 21; 22; 23]. In Table 1, an overview of the hereditary
cancer susceptibility syndrome genes is shown, including their chromosomal location, which syndrome is involved and the clinical features of these syndromes.
Table1 : Summary of the syndromes associated with hereditary breast cancer [ 23]
The remaining familial risk is best explained by a polygenic model in which genetic variants, polymorphisms or mutations in a large number of genes, can contribute together to cancer predisposition even if each one confers a small excess of risk. [ 1; 17; 19]
-Genes involved in breast cancer predisposition
The breast cancer predisposition factors identified to date can be stratified by risk profile into three tiers: high penetrance genes, intermediate-penetrance genes, and low-penetrance alleles. [19]
-High-Penetrance Genes
Mutation in high-penetrance breast cancer predisposition genes confer a greater than tenfold relative risk of breast cancer.
The BRCA1 and BRCA2 genes were discovered in the nineties, starting in 1990 where BRCA1 was for the first time linked to breast cancer using a large group of early onset breast cancer families and linkage analysis. The BRCA1 gene was mapped to chromosome 17q21. In 1994, the BRCA1 gene was cloned and truncating mutations were identified in the coding sequence of the BRCA1 gene in families with multiple cases of breast cancer [ 24 ]. Search for more genes that might be involved in these hereditary susceptibility breast cancer families led in 1995 to the discovery of the BRCA2 gene. The BRCA2 gene is located on chromosome 13q12.3, and was discovered also by linking analysis and positional cloning using familial breast cancer pedigrees in successive generations [25 ]. At the same time, families with high frequencies of male breast cancer were found to carry the BRCA2 mutation [26]. Carriers of the BRCA1 and BRCA2 mutations do not only develop breast cancer and ovarian cancer but also bear an increased risk for developing Fallopian tube, colon, melanoma, prostate and pancreatic cancer [ 27; 28] Both BRCA genes bear rather complex genomic structures. BRCA1 has a highly conserved zinc-binding RING finger domain which is located close to the amino-terminus. RING finger domain proteins are recognized as E3 ligase enzymes that participate in ubiquitination [110]. Mutations in the RING finger domain inactivate BRCA E3 ligase and have an effect on the other tumor suppressor activities of BRCA1 [ 29 ]. Towards the carboxyl terminus of BRCA1 two tandem copies of the same motif are found, designated the BRCT domains. These BRCT domains are regions reported to activate transcription
when fused to a DNA binding domain [30 ]. BRCA2 contains a number of recognizable motifs, the eight copies of a 20–30 amino acid repeat, termed BRC repeats and the ssDNA binding region. Their function is to bind to RAD51 to regulate DNA repair (Fig. 2) [ 31 ].
Fig.2 :Schematic representation of BRCA1 and BRCA2 functional domains and selected binding partners.
TP53 (tumor protein p53) is a tumor suppressor gene located on chromosome
17p13.1 encoding a nuclear phosphoprotein (p53). TP53 acts as a transcription factor involved in the control of cell cycle progression, repair of DNA damage, genomic stability, and apoptosis. TP53 is constitutionally mutated in the Li-Fraumeni syndrome, an autosomal-dominant predisposition to breast cancer and other forms of cancer . The TP53 gene is more commonly altered in BRCA1 (56%– 100%) and BRCA2 (29%) related breast cancer in comparison with non-BRCA related breast [ 32 ]. In BRCA1 or BRCA2 deficient cells changes were seen at TP53 codons that are not the mutation hotspots. Structural modelling showed that most of these p53 non-hotspot aminoacids are distributed in a region of the protein on the opposite side of the p53 DNA-binding surface. Breast cancers with these TP53 non-hotspot mutations were associated with a significantly better prognosis when compared with
TP53 mutations in conserved or structural domains . Preliminary data suggest that BRCA1 or BRCA2 mutations influence the distribution of the TP53 mutations and the way of carcinogenesis, but additional studies must be performed to support this [ 33]. Therefore mutations in TP53 gene are associated with Li-Fraumeni syndrome, a cancer predisposition syndrome, in which there is a high frequency of early onset breast cancer found in association with sarcomas and childhood cancer of the adrenal cortex, brain and other sites. Li-Fraumeni syndrome is rare and mutations in TP53 are uncommon in non-LiFraumeni breast cancer families. Thus, the attributable risk of TP53 mutations to familial breast cancer is very low. [ 19]
PTEN (phosphatase and tensin homolog), is a tumor suppressor gene located on
chromosome 10q23.3. PTEN encodes for the protein phosphatidylinositol phosphate phosphatase and has multiple and as yet incompletely understood roles in cellular regulation [34 ]. Germline mutations in PTEN can lead to a rare autosomal dominant inherited cancer syndrome, Cowden disease, characterized by a high risk of breast-, thyroid- and endometrial carcinomas and hamartomas [ 22 ].
STK11 (LKB1) (Serine/theronine kinase 11) is a gene located on chromosome
19p13.3 that encodes a serine/ threonine kinase and functions mainly through inhibition of the mTOR pathway. STK11 is mutated in the autosomal dominant condition Peutz-Jeghers syndrome, characterized by perioral pigmentation and hamartomatous polyposis [ 35 ]. Patients with this syndrome have a 30%–50% risk of developing breast cancer [ 36 ] .
CDH1 (Cadherin 1, E-cadherin) is a gene located on chromosome 16q22.1 encoding
E-cadherin, a calcium dependent cell adhesion glycoprotein, which is important for cell-to-cell adhesion . Familial diffuse gastric cancer, an autosomal dominant cancer syndrome is caused by mutations in the CDH1 gene and affected women are
predisposed to lobular breast cancer. Patient with a familial diffuse gastric cancer have a risk of about 50% of getting breast cancer [ 37 ] .
Intermediate Penetrance genes
Mutations in these genes are rare and confer a relative risk of breast cancer of 2 to 4. The rarity of mutations and modest associated risks are such that the attributable risk of mutation in these genes is low. Together they account for approximately 2.3 % of excess familial risk. [19]
CHEK2 (checkpoint kinase 2) is a gene located on chromosome 22q12.1 and
encodes a cell cycle checkpoint kinase which is a key mediator in DNA damage response. Mutations in CHEK2 were originally thought to result in the Li-Fraumeni syndrome or in a Li-Fraumeni-like syndrome. The CHEK2 gene has been proposed to be a low penetrance breast cancer susceptibility gene. The 1000delC variant results in an approximately two fold risk of breast cancer in women and a ten fold risk in men. In these cases, there is no mention of co-existence of BRCA1 and BRCA2 mutations. So far, beside the 1000delC and 1157T mutations, no additional CHEK2 mutations have been found [ 38].
ATM (ataxia teleangiectasia mutated) is a gene located on chromosome 11q22.3 and
encodes a checkpoint kinase that plays a role in DNA repair. Biallelic mutations in this gene are linked to the rare human autosomal recessive disorder called ataxia teleangiectasia (AT) , causing a variety of somatic disorders. A heterozygous mutation of ATM does not lead to the AT phenotype but carriers have a two to five fold risk of breast cancer [ 39 ] .
RAD50 is a member of the conserved MRN complex (MRE11, RAD50 and NBS1)
that interacts with BRCA1 and plays a central role in DNA repair. A truncating mutation in RAD50, 657delT, was demonstrated in 8/317 consecutively ascertained
breast cancer cases and 6/1000 controls from Finland. Other rare truncating mutation have been reported in RAD50, but the contribution of RAD50 to breast cancer predisposition outside of Finland requires further clarification. [40]
NBS1 is a gene located on chromosome 8q21 and involved in the Nijmegen breakage
syndrome, a chromosome instability syndrome. Proteins of the gene NSB together with proteins of the genes RAD50 and MRE11, form the the MRN complex involved in the recognition and repair of DNA double strand breaks. The estimated prevalence of the most common mutation is very low and the breast cancer risk conferred by a NBS1 mutation is estimated to be low [ 41].
A rare recessive repair defect disorder called Fanconi anaemia (FA) is linked to a number of genes, in total 12, that, together with BRCA1, are involved in homologous recombination DNA repair mechanisms [ 42 ]. Mutations in FANCJ (=BRIP1), a DEAH helicase that interacts with BRCA1and has BRCA1-dependent roles in DNA repair and checkpoint control, and FANCN (= PALB2) that promotes the localization and stability of BRCA2, are associated with a two fold increased risk of breast cancer [ 43; 44 ]. The remaining ten FA genes may be involved in the carcinogenesis of breast cancer but their role has not been elucidated yet. It has been suggested that these genes are inactivated through epigenetic/transcriptional mechanisms. For example, the FANCD2 protein is down regulated in sporadic and in hereditary breast carcinomas [ 45 ].
Identification of new Breast Cancer predisposition genes
There is a clear discontinuity in the risk associated with the three categories of breast cancer predisposition factors identified to date: the high-penetrance genes confer a risk that is elevated > 10 fold, the known intermediate genes 2-4 fold, and the low-penetrance alleles < 1.5 fold. [ 19] More than 70% of genetic predisposition to breast cancer remains unaccounted for , but new factors may be identified considering how
technological and intellectual advances may allow extension of the repertoire of breast cancer predisposition factors.
Segregation analysis generated no evidence for further dominant genes of a risk-penetrance profile comparable to BRCA1 or BRCA2 and this has been corroborated by linkage studies.
But futher intermediate-penetrance breast cancer predisposition genes exist, although it is difficult to predict how many there may be and what proportion of the total excess familial risk is attributable to them. The known intermediate-penetrance genes are all involved in DNA repair and function in pathways with BRCA1 and/or BRCA2 so genes involved in DNA repair and other relevant pathway represent plausible candidates and are the most intensely investigated. However, technological advances are facilitating increasingly high-throughput mutational screening such as the genome-wide resequencing. This will allow interrogation of regions of the genome not previously investigated and may reveal further intermediated-penetrance genes with functions in pathways not predictable from current paradigm. Large , collaborative epidemiology studies of rare syndrome can optimize power and minimize bias and may represent the optimal strategy by which to establish accurate estimates of these breast cancer risk. Epidemiological studies of recessive syndrome , particularly those associated with childhood cancer, may reveal elevated frequency of breast cancer in relatives of affected individuals and may lead to the identification of further intermediate-penetrance breast cancer genes, as in the case of ATM.
Genome-wide association data offer clear evidence that many further common low-penetrance breast cancer predisposition variants exist but comprehensive study of this extensive repertoire of low-penetrance alleles and larger genome-wide association experiments are required. It is currently unclear what proportion of common low-penetrance alleles will be detectable by feasible study in the immediate future. Strongly associated SNPs (from genome wide scans ) may be utilized for genetic epidemiologic analysis and clinical risk estimation , but the way in which these
common variants contribute to cancer is largely unknown and exploration of the biological mechanism that underlie these signals offers new avenues of study.
- BRCA1 gene
BRCA1 functional domains
BRCA1 is located on chromosome 17q21 and encodes a protein composed of 1863 aminoacid residues. Study of the protein’s structure and comparison to its murine counterpart has helped to highlight some regions that are likely to have functional importance. Homology between human BRCA1 and its murine counterpart is only 58%, a low similarity, suggesting rapid evolution of this gene and protein. Highly conserved regions comprise: a ring finger (RING) domain at the N-terminal region, an acidic region at the C-terminal region, and two putative nuclear localisation signals.[1; 46] The varied roles of BRCA1 are partly dependent on the E3 ubiquitin ligase enzymatic activity that BRCA1 acquires by binding to the protein BARD1. The interaction between BRCA1 and BARD1 occurs through the N-terminal RING domain in each of the molecules.[47] In model organism, mutants of the BRCA1 or BARD1 orthologs exhibit identical phenotype.[48] In human , several cancer-associated BRCA1 mutation alter residues in the RING domain, leading to the loss of E3 ligase activity. [49 ] Mammalian cells contain a large repertoire of E3 ligases, each with a different , but redundant, specificity for target protein. The identity of the functionally relevant intracellular substrates for ubiquitin conjugation by the BRCA1-BARD heterodimer remains elusive. This represents an important gap in our understanding of BRCA1 function. In vitro, the heterodimer can modify a variety of substrates ranging from RNA polymerase II to p53 to the histones H2A, H2AX or H2B to the FA protein FANC2. The fact that most of these substrates continue to be modify in vivo in cells lacking BRCA1 suggests either that they are not authentic targets for this enzymatic activity or that alternative enzymes are able to stand in. [50] The interaction of Abraxas with BRCA1 highlights an important motif in BRCA1 that mediates several of its protein-protein interactions. This is the BRCT domain , a region of ~100 residues that is repeated twice at the C terminus of BRCA1. The BRCT motifs in BRCA1 bind directly to phosphopeptide substrates with the
consensus Ser(P)XXPhe. Protein involved in DNA repair as BRIP, DNA helicase mutated in FA-J and CtIP (carboxy-terminal-binding-protein interacting protein) bind the BRCT of BRCA1. The interaction of BRCA1 with Abraxas, BRIP1 or CtIP via the BRCT domain appear to be mutually exclusive, suggesting the existence of multiple, distinct cellular complexes that may serve distinct function at sites of DNA breakage. Most tumorigenic BRCA1 lesions described in hereditary breast cancer families have mutations or deletions in one or both BRCT domains, suggesting that they have a key role in BRCA1 mediated tumour suppression.[51; 52] BRCT domains are found in many proteins involved in DNA-repair pathways, and bind to many proteins, including RNA polymerase II, p300, BACH1, histone deacetylases (HDACs) 1 and 2, p53, CtIP and RB. The RNA polymerase II holoenzyme binds to both the amino and carboxyl termini of BRCA1; amino-terminal binding is through the BARD1–BRCA1 complex. RAD51 and BRCA2 also bind BRCA1, and the three proteins colocalize in sub-nuclear foci. [53]
p53, MYC, RB and ZBRK1 all bind to a region of BRCA1 that includes the nuclear localization signals (NLSs). ZBRK1 is a zinc-finger protein that suppresses transcription through an interaction with GADD45. BRCA1 is required for this repression. SW1/SNF binding occurs between amino acids 260 and 553. The binding domain encompasses amino acids 452–1,079. It contributes to the DNA-repair-related functions of BRCA1, which are partly mediated through proteins that make up the BRCA1-associated surveillance complex (BASC). Several proteins (including MRE11, RAD50, NBS1, MDC1, ATM, CHK2 and CDK2) bind to the central region of BRCA1. SQ sequences (clusters of serine and threonine sequences), known as SQ-cluster domains (SCDs) are preferred sites of ATM phosphorylation. [53]
BRCA1 expression is associated with cell cycle phases in both normal mammary epithelial cells, and in breast and ovarian cancer cell lines, with expression being induced maximally just before cell entry into S phase, at the time when maximal growth and differentiation is occurring. BRCA1 hyperphosphorylation correlates with
changes in BRCA1 protein concentrations, alteration in subcellular localisation, and changes in mRNA concentrations. This supports the hypothesis that BRCA1 functions as a cell cycle regulator at the G1/S phase transition and/or at the G2/M phase transition.[54]
Fig.3: BRCA1 functional domains and its binding partners
Functions of BRCA1 - Transcription
The concept of BRCA1 as a transcription factor was first suggested by transient transfection assays in which a reporter gene was activated by the carboxyl terminus of BRCA1 fused to the GAL4 DNA-binding domain [ 55 ]. The transcription function of BRCA1 was established by the association of BRCA1 with RNA polymerase II in a large complex called the RNA polymerase II holoenzyme (holo-pol) [ 56 ]. BRCA1 doesn’t function as an enhancer-binding protein, since it does not bind to DNA with sequence-specificity but it binds to DNA independently of sequence with a preference for abnormal structure, even if this is more consistent with a role in DNA repair rather than transcription. The amino-terminal RING-finger domain provides the primary binding to the holo-pol, probably via its association
with BARD1 [ 57 ]. The carboxyl terminus of BRCA1 also binds to the holo-pol via its association with RNA helicase A [ 58 ]. The internal portion of BRCA1 binds to a large number of enhancer-binding factors and so BRCA1 bridges the specific enhancer-binding factor to the holo-pol complex. Finding target genes regulated by BRCA1 overexpression in tissue culture might give insight into genetic pathways abnormally expressed in cells that have mutated BRCA1. Several such experiments have been done using microarray technology [ 59; 60 ]. For example, BRCA1 synergistically stimulated cell cycle arrest and DNA-damage repair genes normally upregulated by p53 (such as p21 and p53R2). p53, in fact, is stabilized by overexpression of BRCA1[ 59 ], particulary BRCA1 stimulates those p53 pathways that lead to survival and repair, rather than to apoptosis. By contrast, PIG3, PERP and Bax, genes involved in p53-dependent apoptosis, were downregulated by BRCA1 overexpression [ 59]. Knocking down BRCA1 expression using antisense RNA leads to an increased sensitivity of cells to apoptosis [59]. Interestingly, BRCA1 does not always save cells from imminent death, in fact overexpression of BRCA1 leads to the up-regulation of genes necessary for apoptosis via the interferon-g pathway [ 61 ]. Therefore, similar microarray studies, using knockdown of BRCA1 by small interfering RNA, might be able to identify genes involved in BRCA1-associated cancer.
-Chromatin remodelling
BRCA1 regulates many types of genes, interacts with transcriptional repressors and activators, and it is also part of the transcriptional machinery. One way to link these roles is to hypothesize that BRCA1 controls chromatin structure. It has been shown that it interacts with the chromatin remodelling factors SWI/SNF and BRG1 [ 62 ] and with histone deacetylase [ 63].
Functional assays showed that full-length BRCA1 tethered to heterochromatin resulted in a partial decompression of the DNA, and , in particular, the isolation of the BRCT domain showed that it vastly reversed the compression of the
heterochromatin. Probably this remodeling function is negatively regulated by the rest of the BRCA1 protein [ 64]. The flip side of this finding was observed for a role in the establishment of heterochromatin by BRCA1. Mutation, or loss of function, of BRCA1 results in an altered phenotype of X chromosome inactivation , a process by which a major heterochromatin domain is established over one X chromosome. XIST is an RNA molecule that coats the inactive X chromosome in female cells and is central to the process by which the entire chromosome is repressed. When BRCA1 is not present in a cell, XIST RNA fails to localize to the X chromosome. The presence or absence of functional BRCA1 does not affect the level of the XIST transcript, just its localization and effectiveness in silencing. Other heterochromatin markers such as macro-histone H2A1 and H3 methylated at Lys9, also failed to localize to the inactive X chromosome without functional BRCA1 [ 65 ].
- DNA damage repair
BRCA1 co-localizes with macro-H2A1 and H3mK9 (histone H3 methylated at Lys9) at the inactive X; it has also been found to co-localize with another modified histone, phosphorylated H2AX (g-H2AX), after DNA damage . Unphosphorylated H2AX is interspersed in chromatin throughout the genome, and after DNA damage, one of the earliest events is the phosphorylation of Ser139 of H2AX. g-H2AX forms discrete foci within 10 minutes of DNA damage with BRCA1 detectable within 30 min in some cell lines, or within six hours in others. After BRCA1, either RAD50 or RAD51, but not both, co-localize with DNA-damage-induced foci [ 66]. Not surprisingly, H2AX-null cells are hypersensitive to ionizing radiation and have increased spontaneous chromosomal aberrations and the formation of BRCA1 and RAD51 DNA-damage-induced foci were decreased [ 67].
BRCA1 regulates a variety of DNA-damage-repair pathways. BRCA1-deficient cells have defects in transcription-coupled repair, homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology end-joining [68;69; 70; 71]. In vitro binding assays have also revealed that BRCA1 binds to several factors
involved in the repair pathways. The diversity of the repair mechanisms is suggestive of an indirect effect of BRCA1 on these various pathways. BRCA1 could transcriptionally activate the genes encoding the repair-effector enzymes or, alternatively, some other activity such as ubiquitination that might regulate the effector proteins.
-Ubiquitination
The RING-finger domain, such as is found at the BRCA1 amino terminus, is commonly associated with ubiquitin ligase activity. In this process a ubiquitin ligase polymerizes ubiquitin on a target protein and then the ubiquitinated target is degraded by the proteasome. This process is quite specific, in fact specific polypeptides in a protein complex may be ubiquitinated and degraded, leaving behind the other subunits intact [ 72 ]. BRCA1, together with BARD1,forms a heterodimer associating via the RING-finger domains and adjacent α-helices, and functions as an active ubiquitin polymerase [ 73]. Like an ubiquitin polymerase, the heterodimer synthesizes long chains of ubiquitin, but the key will be to identify the target for the ubiquitin ligase function. Although ubiquitination is usually linked to protein degradation via the 26S proteasome, BRCA1–BARD1 might also monoubiquitinate proteins, or polymerize ubiquitin via different lysine linkages that do not target the protein to degradation but into some other pathway. BRCA1–BARD1 monoubiquitinates histone monomers, including unphosphorylated H2AX [ 74 ]. Most finger proteins do not specify the ubiquitination target via the RING-finger domain, but rather by some other domain of the same polypeptide, or another subunit of multisubunit ubiquitin ligases. In general, in the current studies only the BRCA1–BARD1 RINGfinger domains are isolated, but BRCA1–BARD1 are large proteins and thus it is likely that the untested 90% of these proteins regulate the ubiquitin ligase enzymatic activity or specify its target. Ideally, using full-length BRCA1–BARD1, specific targets of ubiquitination and functional consequences will be identified in the near future. The RING fingers of BRCA1 and BARD1, which
ordinarily heterodimerize, can form 30 nm ring-shaped superstructures that are visible by electron microscopy. These super-RING structures are more enzymatically active than dimeric BRCA1–BARD1, and the ring structure functions as a scaffold for other proteins to couple reactions. The E2 ubiquitin conjugating enzyme, UbcH5c, forms a circle surrounding the BRCA1–BARD1 complex, and ubiquitin chains dot the surface. This scaffold arrangement renders the ubiquitination activity of BRCA1– BARD1 highly processive [ 75 ]. A potential link of this ubiquitination activity to the transcription function of BRCA1 could be via the recently established role of ubiquitination in transcriptional activation. In fact BRCA1 may ubiquitinate the enhancer-binding factors or alternatively, transcriptional silencing has been observed to be mediated in part via monoubiquitination of histones. [ 76 ].
Fig.4 : A subset of known nuclear interactions by BRCA1 are shown and grouped according to function: (a) Transcription and chromatin remodelling,(b) ubiquitination, and (c) DNA repair.
-
DNA Damage Response and Repair Mechanism
Every day our cells battle against genetic damage that could lead to a variety of genetically inherited disorders, in aging, and in carcinogenesis. DNA repair is a sort of innate insurance policy for the normal daily toil of our genes against damage resulting from cytotoxic and mutagenic effects of DNA damaging agents. Environmental DNA-damaging agents include UV light and ionizing radiation, as well as a variety of chemicals encountered in foodstuffs, or as air- and water-borne agents. Endogenous damaging agents include metabolites that can act as alkylating agents and the ROS (Reactive Oxygen Species) that arise during respiration. DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by these exogenous and endogenous agents and exposure to carcinogens and cytotoxic compounds like inhaled cigarette smoke, or incompletely defined dietary factors. Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and cellular cytotoxicity.
The major forms of DNA damage include SSB (Single-strand Breaks), DSB (Double-strand Breaks), alteration of bases, hydrolytic depurination, hydrolytic deamination of cytosine and 5-methylcytosine bases, formation of covalent adducts with DNA, and oxidative damage to bases and to the phosphodiester backbone of DNA. The vast majorities of these lesions are repaired by [ 77]:
-BER: base excision repair– replaces damaged bases in the DNA code
-NER: nucleotide excision repair– replaces a string of bases if one or more is
damaged
-MMR: mismatch repair - corrects mismatches in the sequence of bases in DNA -NHEJ: non-homologous end joining– fixes double-strand breaks in the DNA
double-helix
-HR: homologous repair- fixes double-strand breaks in and interstrand cross-links in
BER involves the concerted effort of several repair proteins that recognize and excise
specific DNA damages, eventually replacing the damaged moiety with a normal nucleotide. BER has two subpathways, both of which are initiated by the action of a DNA Glycosylase. Such DNA Glycosylases bind specifically to a target base and hydrolyze the N-glycosylic bond, releasing the inappropriate or damaged base while keeping the sugar phosphate backbone of the DNA intact. This cleavage generates an AP (Apyrimidinic/Apurinic) or a basic site (i.e. the site of base loss) in the DNA. The AP site is subsequently processed by APE1 (AP Endonuclease-1, also called HAP1/REF1/APEX) which cleaves the phosphodiester backbone immediately 5' to the AP site, resulting in a 3' hydroxyl group and a transient 5’ dRP (abasic Deoxyribose Phosphate) [ 78 ]. Removal of the dRP is accomplished by the action of DNA Pol Beta (Polymerase Beta), which adds one nucleotide to the 3’ end of the nick and removes the dRP moiety via its associated AP lyase activity. A DNA Ligase seals the strand nick, thus restoring the integrity of the DNA. Replacement of the damaged base with a single new nucleotide is referred to as short-patch repair and represents approximately 80-90% of all BER. DNA Pol Beta also interacts with XRCC1, which acts as a scaffold protein by bringing the polymerase and ligase together at the site of repair and stabilizing the complex [ 79 ]. In cases where the terminal sugar-phosphate residue is resistant to cleavage by the AP lyase function of DNA Pol Beta, DNA strand displacement occur, involving either DNA Pol Beta or a larger polymerase such as DNA Pol Delta for filling-in of gaps a few nucleotides long. This represents the back-up pathway of BER, termed long-patch repair. PARP (Poly ADP Ribose Polymerase), which binds to and is activated by DNA strand breaks, is important when BER is initiated from a SSB to protect and trim the ends for repair synthesis. The FEN1 (Flap Endonuclease-1) structure-specific nuclease removes the displaced dRP as part of a "flap" oligonucleotide prior to sealing of the nick by DNA Ligase and the PCNA (Proliferating Cell Nuclear Antigen) protein stimulates these reactions [80 ], acting as a scaffold protein in this alternative pathway in a way similar to that of XRCC1 in the main pathway.
Fig.5: BER mechanism
NER is perhaps the most flexible of the DNA repair pathways considering the
diversity of DNA lesions it acts upon. The most significant of these lesions are pyrimidine dimmers caused by the UV component of sunlight. The NER process involves damage recognition, local opening of the DNA duplex around the lesion, dual incision of the damaged DNA strand, gap repair synthesis, and strand ligation. There two distinct forms of NER: GG-NER (Global Genomic-NER), which corrects damage in transcriptionally silent areas of the genome, and TC-NER (Transcription Coupled-NER), which repairs lesions on the actively transcribed strand of the DNA. In GG-NER, the XPC (Xeroderma Pigmentosum Complementation Group-C) protein complex is responsible for the initial detection of damaged DNA. Conversely, damage recognition during TC-NER does not require XPC, but the stalled RNA Polymerase complex is then displaced in order to allow the NER proteins to access the damaged DNA. This displacement is aided by the action of the CSA (Cockayne Syndrome-A), CSB proteins and XAB2 (XPA Binding protein-2), as well as other TC-NER-specific factors. The subsequent steps of GG- and TC-NER proceed in an
essentially identical manner. XPA and the heterotrimeric RPA (Replication Protein-A) bind at the site of injury ; next, the XPB and XPD Helicases, arrive in the immediate vicinity of the lesion and the endonucleases XPG and ERCC1 (Excision Repair Cross-Complementing group-1)/XPF cleave one strand of the DNA at positions 3' and 5' to the damage, respectively, generating an approximately 30 base oligonucleotide containing the lesion. This oligonucleotide is displaced, making way for gap repair synthesis (performed by DNA Pol Delta/Epsilon, as well as several replication accessory factors). Finally, DNA Ligase seals the nick in the repaired strand, thus completing the NER process [ 81 ]. Three syndromes are associated with inborn defects in NER: XP, CS and TTD (Trichothiodystrophy), all characterized by exquisite sun sensitivity [ 82 ].
Fig.6 : NER mechanism
The MMR system is responsible for the post-replicative repair of mismatches and small single stranded DNA loops, and it is critically involved in preventing
recombination between homologous DNA sequences. MMR removes nucleotides mispaired by DNA Polymerases and IDLs (Insertion/Deletion Loops) that result from slippage during replication of repetitive sequences or during recombination. Defects in this system dramatically increase mutation rates, fuelling the process of oncogenesis. Four principal steps involved in MMR are: (1) recognition of base-base mismatches and IDLs; (2) recruitment of additional MMR factors; (3) search for a signal that identifies the wrong (newly synthesized) strand, followed by degradation past the mismatch; and (4) resynthesis of the excised tract [ 83 ]. The eukaryotic MMR system involves two different heterodimeric complexes of MutS-related proteins, MSH2-MSH3 (known as MutSBeta) and MSH2-MSH6 (known as MutSAlpha), and each has different mispair recognition specificity. Heterodimers MSH2-MSH6 focuses on mismatches and single-base loops, whereas MSH2-MSH3 dimmers (MutSBeta) recognize ILDs [ 84 ]. Excision and resynthesis of the nascent strand (containing the mismatch or IDL) is performed by a number of proteins including PCNA, RPA, RFC (Replication Factor-C) Exonuclease-I, DNA Polymerases Delta/Epsilon, FEN1, and additional factors. MMR has an important role in genetic recombination beyond the repair of mispaired bases by regulating the resolution of Holliday junctions and is involved in cancer especially HNPCC (Hereditary Non-Polyposis Colorectal Carcinoma) that exhibit widespread alterations of poly (A) tracts [84].
DSBs are the most serious form of DNA damage because they pose problems for
transcription, replication, and chromosome segregation. DSBs affect both strands of the DNA duplex and therefore prevent use of the complementary strand as a template for repair. Cells have evolved two distinct pathways of DSB repair, HR
(Homologous Recombination) and NHEJ (Non-Homologous End Joining). HR-directed repair corrects DSBs defects in an error-free manner using a mechanism
that retrieves genetic information from a homologous, undamaged DNA molecule. The majority of HR-based repair takes place in late S- and G2-phases of the cell cycle when an undamaged sister chromatid is available for use as repair template. The Rad52 epistasis group of proteins, including Rad50, Rad51, Rad52, Rad54, NBS1 (Nijmegen Breakage Syndrome-1) and MRE11 (Meiotic Recombination-11) function in the initial steps of meiotic recombination and are also involved in recombination processes in mitotic cells [ 85 ]. The Rad52 protein itself is the initial sensor of the broken DNA ends. Processing of the damaged ends ensues resulting in the production of 3' ssDNA overhangs. The newly generated ssDNA ends are bound by Rad51 to form a nucleoprotein filament. Other proteins including RPA, Rad52, Rad54, BRCA1, BRCA2 and BARD1 , and several additional Rad51-related proteins serve as accessory factors in filament assembly and subsequent Rad51 activities. The Rad51 nucleoprotein filament searches the undamaged DNA on the sister chromatid for a homologous repair template. Once the homologous DNA has been identified, the damaged DNA strand invades the undamaged DNA duplex in a process referred to as DNA strand exchange. A DNA Polymerase then extends the 3' end of the invading strand and subsequent ligation by DNA Ligase-I yields a heteroduplexed DNA structure. This recombination intermediate is resolved and the precise, error-free correction of the DSB is complete.[85] In the NHEJ, where the two DNA ends are connected without the need for longer stretches of homology, repair of a DSB is error prone and frequently leads to small deletions. NHEJ rejoins the two broken ends directly and generally leads to small deletions of DNA sequence. The activity of the Ku70/Ku80 heterodimeric protein is essential for NHEJ. The Ku heterodimer initiates
NHEJ by binding to the free DNA ends and recruiting other NHEJ factors such as DNA-PKcs (DNA-dependent Protein Kinase), XRCC4, XRCC7 and DNA Ligase-IV. In addition to the Ku and Ligase-IV homologs, the Rad50, MRE11 and NBS1 genes are also involved in NHEJ, particularly if the DNA ends require processing before ligation. Unrepaired DSBs can be lethal, whereas misrepaired DSBs can cause chromosomal fragmentation, translocations and deletions. Such lesions are potential inducers of carcinogenesis through activation of proto-oncogenes, inactivation of tumor suppressor genes or loss of heterozygosity [85].
Many of the checkpoint proteins are activated in response to DNA damage, and a number of phosphorylation events in DNA repair mechanism depend on activation of kinases. The ATM (Ataxia Telangiectasia-Mutated)/ATR (ATM/Rad3-related) family act early in the checkpoint pathways either as DNA-damage detectors or in close association with such detectors. In mammalian cells, these kinases are activated in a lesion-specific fashion, with ATM being specific for agents that induce DNA-DSB, and ATR, probably responding to UV- induced damage. Additional conserved checkpoint components that function in concert with the ATM/ATR kinases includes the "checkpoint Rad" proteins Rad1, Rad9, Rad17, Rad26, and Hus1, loss of any one of which confers radiation sensitivity and checkpoint defects. Specifically, Rad1, Rad9 and Hus1 are related to the DNA Polymerase accessory protein PCNA, whereas Rad17 is related to subunits of RFC. During DNA replication RFC functions to load PCNA onto chromatin. Just as PCNA acts by tethering replicative polymerases to their template, Rad1/Rad9/Hus1 form a sliding clamp, which has the dual purpose of checkpoint signaling and recruiting DNA repair enzymes [86 ].
Fig.8: Mechanisms of a) HR and b) NHEJ
DNA-damage checkpoints in higher eukaryotes have acquired additional levels of complexity. The tumor suppressor p53 is central to these higher eukaryotic checkpoint controls in DNA damage-induced G1 arrest through transcriptional induction of the cyclin-dependent kinases Chk1 and Chk2, and inhibitor p21 (WAF1), which binds to and inhibits G1 cyclin-dependent kinases. Activation of p53 in response to DNA damage and other stresses involves complex post-translational modification of p53 and its negative modulator MDM2 (Mouse Double Minute-2), which is itself induced by p53 at the transcriptional level. MDM2 targets p53 for proteolysis by the ubiquitin/proteasome pathway, and downregulation of this proteolytic pathway following DNA damage allows p53 accumulation. GADD45 (Growth Arrest and DNA Damage-inducible) is one of several known p53 target genes, which is involved in a variety of growth regulatory mechanisms, including DNA replication and repair, G2/M checkpoint control, and apoptosis through
activation of JNK (c-Jun N-terminal Kinase)/SAPK (Stress-Activated Protein Kinase) pathway. It binds to several proteins involved in these processes, including PCNA, p21 (WAF1), and CDC2 (Cell Division Cycle-2) [87 ].
An increasing number of human hereditary diseases that are characterized by severe developmental problems and/or a predisposition to cancer have been linked to deficiencies in DNA repair.
- BRCA1 and Homologous Recombination Mechanism
Homologous Recombination (HR ) has essential roles in meiosis and mitosis. In meiosis, HR mediates the exchange of information between the maternal and paternal alleles within the gamete precursor cell and thus generates diversity among the progeny derived from common parents. HR has a second critical function in fact it ensures proper segregation of homologous chromosome pairs at the first meiotic division through the formation of crossover, resulting in gametes with the correct number of chromosomes. HR maintains somatic genome stability by promoting accurate repair of DSBs induced by ionizing radiation and other agents, repair of incomplete telomeres that arise when the enzyme telomerase is non-functional, repair of DNA interstrand crosslinks, and the repair of damaged replication forks. Although cells have alternative DNA repair pathway such us the Non Homologous End-Joinig, these may not be operative at all phases of the cell cycle, they do not always act on injured replication forks, nor are they as precise as HR in the repair of broken chromosomes. Mutations in genes encoding the enzymatic step of HR result in extreme sensitivity to DNA-damaging agents such as ionizing radiation in model organisms. In fact many HR genes were first identified by mutants that are hypersensitive to DNA-damaging agents that cause DSBs and by a failure to give viable meiotic product.[88; 89]
The model proposed for the double-strand break repair by homologous recombination [ 90; 91 ] provides different steps:
a) A DSB in the DNA generates regions of ssDNA. This step is promoted by the RAD50/MRE11/NBS1 complex associated with CtIP in mammals .
b) ssDNA is covered by the RPA (Replication protein A) protein.
c) RAD52 (in yeast) or BRCA2 (in mammals) displaces RPA from the ssDNA and loads the key protein for HR, RAD51.
d) The ssDNA-RAD51 complex finds the intact homologous double-stranded DNA and promotes the exchange of identical strands and the hybridization of complementary strands.
e) DNA polymerase fills in the gap and moves the displacement loop (D-loop). f) The cruciform junctions (Holliday junctions) are then formed.
g) The resolution of the Holliday junctions depends on the direction of resolution and can proceed via the following two mechanisms: without crossing over or with crossing over (exchange of adjacent DNA sequences).
Fig.9 :Model of double-strand break repair by homologous recombination
Genetic studies conducted in BRCA1- and BRCA2-defective cell lines [ 92] have further revealed that these tumour suppressor genes are required for maintenance of genome integrity and for normal levels of resistance to DNA damage. Moreover, without functional BRCA genes, cells are inefficient in repairing DNA damage by homologous recombination [93]
Studies on BRCA1 and BRCA2 led to the conclusion that the two proteins function together as part of a complex that acts directly in double-strand break (DSB) repair. First, BRCA1- and BRCA2-defective cell lines and knockout mice exhibit similar sensitivities to DNA-damaging agents [94 ]. Second, BRCA1- and BRCA2-defective cell lines show defects in DSB repair by homologous recombination [95]. Third, the BRCA1 and BRCA2 proteins co-localize to DNA damage-induced nuclear foci, and to foci that appear in S phase [96]. But the functions of BRCA1 in DNA repair are not restricted to HR. Instead, BRCA1 performs varied functions in the cellular response to DNA breakage or replication arrest, on the other hand serving locally at sites of DNA damage to recruit molecules that sense and repair DNA lesions, and on the other as an effector of response to DNA damage. In contrast, BRCA2 works largely at sites of DNA repair as a specific mediator of the reaction that lead to HR. [1 ]
When we speak of the role of BRCA1 in the repair mechanisms, we necessary mention the BASC complex (BRCA1-Associated Genome Surveillance Complex), a super complex of BRCA1 with a key role in the recognition and repair of DNA damage. This complex includes tumor suppressors and DNA damage repair proteins as MSH2, MSH6, MLH1, ATM (Ataxia-Telangiectasia), BLM (Bloom syndrome), and the Rad50-MRE11 (Meiotic Recombination-11)-NBS1 (Nijmegen Breakage Syndrome) protein complex. [97; 98]
Fig.10: Interactions of BRCA1 and BRCA2 with other proteins, or protein complexes. as
determined by two-hybrid screens, immunoprecipitation analyses and co-fractionation studies
BRCA1 participates in HR interacting with the RAD51 recombination enzyme and BRCA2 [99] which work directly in the reaction that lead to HR. However, the interaction between BRCA1 and BRCA2-RAD51 may not be direct, but may be bridged by other molecules. How BRCA1 might regulate BRCA1 or RAD51 in HR reactions is therefore unclear. Control via ubiquitin modification is possible, but at moment there aren’t supporting evidence reported.
Fig.11: Distinct complexes formed by BRCA1 and BRCA2 are involved in a variety of cellular processes such as damage signalling, protein degradation and both direct and indirect effects on the mechanisms of homologous
recombination and DNA repair
The ATM and ATR (ATM and Rad3 related) kinases, both implicated in responses to genotoxic stress, are also involved for the radiation-induced phosphorylation of BRCA1 [100]. Normally, ATM phosphorylates Chk2 (Chk1 Checkpoint Homolog), which in turn phosphorylates BRCA1. The ring finger of
BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another ring-containing protein, BARD1 (BRCA1 Associated Ring Domain-1), and is required for the function of this tumor suppressor protein in protecting genomic integrity [101]. ATR and ATM kinase targets also include repair enzymes like Rad51, Chk1 and Chk2. In response to ionizing radiation, ATM phosphorylates NBS1 leading to phosphorylation of FANCD2 and the establishment of an S-Phase checkpoint response, and in response to Mitomycin-C or Hydroxyurea, NBS1 assembles in nuclear foci with MRE11-Rad50 and FANCD2. Like ATM, the MRE11 complex is a crucial upstream regulator of checkpoint responses and DNA-repair responses in all eukaryotic cells. The MRE11 complex assembles with BRCA1 in nuclear foci following DNA damage and regulate homologous recombination repair [102].
There are mounting evidence that BRCA1 works locally at the sites where these lesions occur [103], in fact it is an early migrant to sites of H2AX phosphorylation [104], suggesting a proximal role in the events that follow at the damage site. The RAP80 protein is required for recruitment, and it binds indirectly to BRCA1 via another protein, Abraxas. RAP80 contains multiple motifs that mediate its interaction with ubiquitin moieties, which are responsible for its localization to site of DNA breakage. Thus, the tri-molecular complex between BRCA1, Abraxas, and RAP80 engages ubiquitin-conjugated substrates at breakage sites. What initiates ubiquitinationat these sites is uncertain, but once recruited, the BRCA1-BARD1 enzyme may further propagate this modification to other substrates in the vicinity. [52]
BRCA1 co-localized in the subnuclear foci with Rad51 and with FANCD2 (Fanconi Anemia subtype D2 protein), witch is monoubiquitylated by other DNA damage activates [105]. FA (Fanconi Anemia) is genetically heterogeneous, with at least eleven or more genetically distinct groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG and FANCL), each caused by recessive mutations in a different gene [52; 105]. Five of the proteins (FANCA, FANCC,
FANCE, FANCF and FANCG) assemble in a multisubunit nuclear complex required for the activation of FANCD2 to a monoubiquitinated isoform (FANCD2-Ub), either in response to DNA damage or during S-Phase of the Cell Cycle, thereby targeting FANCD2 to DNA repair nuclear foci containing BRCA1, BRCA2 and Rad51, which are important in maintaining genomic stability by promoting homologous recombination repair [106].
Other protein involved in DNA repair as BRIP, DNA helicase mutated FA-J and CtIP protein bind to the BRCT domain of BRCA1 (with the consensus Ser(P)XXPhe), but the interactions of BRCA1 with Abraxas, BRIP1 or CtIP via BRCT domain appear to be mutually exclusive. This suggests the existence of multiple, distinct cellular complexes that may serve distinct functions at site of DNA breakage.[52] A function of the interaction of CtIP, as well as the MRE11/RAD50/Nbs1, with BRCA1, may be in the resection of flush DSB ends to generate single-stranded DNA tracts that are necessary to initiate HR. Moreover, because single-stranded DNA is a trigger for the activation of checkpoints that arrest cell-cycle progression during the S phase or at the G2/M boundary, we can also speculate this role for BRCA1. [52; 107]
BRCA1 has also been implicated in gene regulation. It induces GADD45, a p53-regulated and stress-inducible gene that plays an important role in cellular response to DNA damage. BRCA1 activation of the GADD45 promoter is mediated through the OCT1 and CAAT motifs located at the GADD45 promoter region [108]. BRCA1 can trigger a G1 arrest that is mediated by transcriptional activation of p21Waf1/Cip1. In addition to its association with holoenzyme, BRCA1 can bind to several different transcription factors, including p53, Myc, STAT1, and CtIP (CBP-Interacting Protein) [109]. BRCA1 acts in concert with STAT1 to differentially activate transcription of a subset of IFN-Gamma target genes and mediates growth inhibition by this cytokine. BRCA1 also binds preferentially to the hypophosphorylated form of Rb (Retinoblastoma Protein) [110].
The BRCA1 RING finger associates with ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family and leads
to transcriptional activation of ATF1 target genes, some of which are involved in the transcriptional response to DNA damage [111].
BRCA1 may also promote local activities at site of DNA breakage interacting with enzymes that modify chromatin or DNA structure, such as BRIP helicase, SWI/SNF chromatin remodelling complex or regulator of histone acetylation and deacetylation. Perhaps the changes in chromatin or DNA structure mediated by these effectors make DNA lesions more accessible to the repair machinery, but how these interactions may assist HR remain speculative. [107]
It’s difficult to assemble different experimental evidence in a coherent model for the participation of BRCA1 in HR. The simplest generalization is that BRCA1 works proximal to the factors that carry out repair by HR, performing several distinct functions that indirectly regulate the efficiency of the process.
- Pathogenic variants and VUS (Variants of Unknown clinical
Significance)
BRCA1 or BRCA2 germline mutation carriers have a lifetime risk of up to 80% for developing the disease. In addition, BRCA1 mutation carriers also have a lifetime risk of up to 56% for developing ovarian cancer . Therefore, identification of BRCA1 and BRCA2 mutation carriers is important because it provides an opportunity to take preventive measures such as prophylactic chemotherapy or mastectomy to reduce the mortality and agony associated with the disease. To identify BRCA1 and BRCA2 mutation carriers, sequencing- based genetic tests are available [112].
While thousands of BRCA1 and BRCA2 truncating mutations have been associated with increased risk of cancer in carriers, the contribution of other BRCA1 and BRCA2 variants to cancer risk remains largely undefined. A very important open question is the interpretation of the actual risk(s) associated with mutations that do not clearly disrupt the gene and are considered as variants of unknown clinical significance (VUSs). The prevalence of VUSs in the population is high. A study conducted by Myriad Genetics Inc. reported that 13.0% of 10,000 individuals tested had a VUS [ 113].
VUS are mainly missense mutations but also include a number of intronic variants and in-frame deletions and insertions [ 114 ; 115]. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, a subset of which are listed in the open access, on-line Breast Cancer Information Core (BIC) Database (http://research.nhgri.nih.gov/bic/) that functions as a repository of sequence alterations in BRCA1 and BRCA2.
At present, the clinical relevance of relatively few of these variants has been established but some were classified as neutral/ no clinical significance [ 116 ] and a small number of variants located in evolutionarily conserved residues [ 117 ] or splicing consensus sites were classified as deleterious using a statistical genetic combined likelihood model [ 116; 118 ], or in vivo splicing/exon skipping analysis [ 119 ]. Classification of other BRCA1 and BRCA2 variants as cancer predisposing
or neutral has proven problematic because it is not known whether these subtle changes alter the function of the proteins sufficiently to predispose to cancer and there is insufficient information from family studies of these rare variants to allow classification using the likelihood model. In figure 12 are illustrated the missense mutations in BRCA1 by showing the location and frequency of all missense variants listed in the BIC database and by indicating the small number of variants that have been definitively classified as either deleterious or neutral.
Fig.12: Diagram of human BRCA1 with domain and motifs(green box, RING domain; blue circles, BRCT domain; black bars, nuclear localization signal)that reports the number of entries in the BIC database for each missense variant. Variants are show as black(unclassified), red (phatogenic), and blue(neutral) bars.
Therefore, it is imperative to understand the functional significance of such variants. At present, so, there is a multifactorial classification model which combines a number of independent features to establish the likelihood that the VUS has the characteristics of known pathogenic mutations. The evaluation was based on family data including family history and cosegregation with the disease in families; tumor
data including histopathological features and loss of heterozygosity (LOH); VUS features including the frequency in cases and controls, cooccurrence with a deleterious mutation in the same gene, the severity of the nucleotide or amino acid change and its conservation across species, and its effect on RNA splicing. However, the majority of the VUS are rare and so such information are frequently not available. For this reason , different type of functional assays based on complementation by cDNAs in BRCA-deficient cell lines have been developed [120; 121; 122]. Although these can distinguish between neutral and deleterious variants, they are of very limited use. An assay based on the transcriptional activation function of the C-terminal domain of BRCA1 is also frequently used [121]. Because BRCA1 is known to be involved in multiple cellular processes, an assay that can be used to study various aspects of BRCA1 function is much needed.
Functional assays to test the VUS(s)
Since the exact biochemical functions of the multifunctional BRCA1 and BRCA2 proteins are not fully defined, it is not clear which in vitro assays measure functions important in carcinogenesis and which are useful in defining whether a variant is pathogenic or not. It has been necessary to develop multiple assays that evaluate specific functions in cells that are known to be dependent on BRCA1 or BRCA2. In some cases, the resulting assays may be limited to specific domains of these proteins. Candidate functional assays must also be fully evaluated for the ability to discriminate between a series of well-defined positive and negative controls [122 ]. Especially now I focus on the description of some assay used for BRCA1.
-Transcription Activation (TA) assay
Early studies of BRCA1 noted that the carboxy-terminal region of BRCA1 functions as a transactivation domain when expressed as a fusion to a heterologous DNA binding domain (DBD) in yeast and mammalian cells [123 ]. Cancer-associated
nonsense, frameshift, and missense mutations, but not benign variants, in this region of BRCA1 were found to impair this transcriptional activity. This led to the development of a transcription assay based on the notion that it is a reliable monitor of the integrity of the BRCT domain and that it can reliably predict the functional effects of mutations.
The assay can be performed in yeast and mammalian cells using expression vector constructs (pLex9 and pcDNA3, respectively) encoding a DNA binding domain (DBD) fusion to residues 1396−1863 of BRCA1. In yeast, the pLex9-BRCA1 construct is transformed into cells with a lacZ reporter construct driven by a lexA binding sites. Transcriptional activity is measured by a quantitative liquid ß-galactosidase assay [124 ]. In human cells (HEK 293T), the variants in a GAL4 DBD fusion are co-transfected with a luciferase reporter driven by five GAL4 binding sites (pG5luc) [ 123 ]. An internal control of Renilla luciferase normalizes the luciferase activity from the reporter. Protein levels measured by western blot allow adjustment for transfection efficiency and expression levels. In this assay wild type BRCA1 and the S1613G neutral variant can serve as positive controls and the M1775R, Y1853X and A1708E cancer-associated mutations can serve as negative controls in both yeast and mammalian cells [123; 124].
This assay has been used to evaluate over 50 different VUS from the BRCA1 BRCT domains. Efforts to validate the assay have focused on the correlation between the assay results and the results from the likelihood model that is based on family data. While this approach has been limited by the relatively small number of variants from the BRCA1 BRCT domains that have been classified by the likelihood model, a strong correlation appears to exist [125 ].
Overall, while this assay may be an excellent method for evaluating the effects of variants on BRCA1 function, it is presently limited to the C-terminus of BRCA1 (aa 1396−1863) and it is not likely that other regions of the protein can be interrogated using this assay.
