The repair gene BACH1 - a potential oncogene.

Lung cancer is the most commonly diagnosed cancer and it is one of the reasons for cancer death worldwide. Approximately 1.6 million case results in deaths per year. 100 The molecular mechanisms which play the role in malignancy are unknown. The important genes which have a role in lung cancer are the cell cycle and the repair genes like TP53 , RB , BRD7 , PCNA and NFKB1 . 101 BRIP1 is found to be overexpressed in lung cancer (COSMIC data, 2014, Figure 6 ). Homozygous deletions are observed in lung adenocarcinoma in the BRIP1 gene (3%). Also, BRCAness, i.e. HR defects in absence of any germline mutation in BRCA , is usually seen in non-small cell lung cancer (NSCLC). 102 High transcript level expression of BRCA1 is a helpful tool for choosing NSCLC patients for individualized chemotherapy, as it is the only independent prognostic variable for NSCLC patients. 103 The findings of Zhang group highlights that the integrity of the FA-BRCA pathway is a determinant of sensitivity/resistance to DNA crosslinking agents in lung cancer cells and may represent a mechanism underlying the resistance to chemotherapy of DNA crosslinking agents. 104 Ubiquitous type of mutation having BRIP1 variants are identified from tumor and blood sample obtained from NSCLC patients. 105 In lung cancer, germline mutations are observed in the CHK1 gene which is involved in Fanconi anemia and BRCA1/2 signaling pathways. 106 Methylation in FANCF promoter is a significant predictor for poor survival in adenocarcinoma of the lung, so inactivation of FANC-BRCA pathways may result in the poorer survival rate of patients with lung cancer. 107 These findings justify the important role played by FANC/BACH1 in cancer metabolism of lungs.

Our current understanding indicates that BACH1 nuclear protein differentially participate in complex networks that regulate cell growth, cell cycle, DNA replication, DNA repair, mitotic chromatin dynamics, and also epigenetic modifications at the specific heterochromatin sites. BACH1 functions in the replication of the difficult sites and during stress, damage and secondary structures, because of its characteristics as a helicase, repair gene and as chromatin remodeler. Cancer mutation data (COSMIC) shows the widespread mutation of this gene in different cancer types. The overexpression of this gene in different cancer types clearly explains the increase in damage in the process of tumorigenesis and the proper repair activity is highly abrogated leading to accumulation of mutations. High mutation burden provides a favorable environment for the development, progression and recurrence of tumor. Further analysis reveals that the HBB domain of BACH1 is the most affected domain and the hot spot for characterized as well as uncharacterized mutations, explaining its role in cancer biology. Since the HBB domain has no link with BRCA1 interaction, so the effect conferred by this domain in different cancer types is independent of the BRCA1 function. The BRCA1 binding domain or the DNA helicase comes as the second most affected region of BACH1. The analysis of variants with uncertain significance also shows HBB domain as the most susceptible sites in BACH1 which justifies the emerging role of DNA repair through BACH1 in cancer biology. A deep insight into the functional aspect of the HBB domain along with BRCA1 interaction will open new avenues in the treatment of most of the deep-rooted cancers. Mutations or defect in this gene affects major molecular pathways that regulates and maintains the genomic integrity of the cells. With an aberration in the genetic integrity tumorigenesis develops. So, BACH1/BRIP1/FANCJ/ChlR1 gene has high potentials of transforming a normal cell into a tumor cell if compromised under certain circumstances, thus justified to be named as an oncogene. Even-though BACH1 has a substantial role in cancer biology and has a major role to play in different types of cancer, very few studies have been completed towards understanding the mechanism of how the proteins interact among themselves. In few of the cancers, BACH1 is analyzed as the major interactor protein of BRCA1, so, a detailed analysis of the interaction study is required to identify its role in tumorigenesis and metastasis. Current literature and our ongoing studies indicate that BRCA1-BACH1 interaction is lost due to diseased condition or a mutation at the interactor domain results in downregulation of DNA proofreading activity leading to more mutations, and hence increasing the risk of tumorigenesis. So, to understand BACH1 , it is essential to explore this protein, its functional and interacting domains and critically evaluate its involvement to physiology and identify the potential roles in human pathologies, such as cancer.

BACH1/BRIP1 / FANCJ / hCHLR1 ( BRCA1 associated C-terminal helicase 1), which is the homolog of yeast Chl1p helicase, is a phosphoprotein located on chromosome 17q22. 1-3 It consists of 1249 amino acid residues with the protein size of 130 KDa, the gene length of 180kb and contains 20 exons ( Figure 1 ). 1 , 4 , 5 BACH1 helicase is present in both active and inactive forms depending on the phosphorylation status at the K52 position of the protein. The dephosphorylated BACH1 leads to the activation of helicase, which is involved in the timely progression of S-phase, repair of DNA cross-links and secondary structures formed during replication and replication induced stress. 6 , 7 Thus phosphorylateddephosphorylated state of BACH1 plays a major role in cell cycle regulation through activation of various pathways in BRCA1 dependent and independent manner. 4 During replication stress, it acts with DNA topoisomerase-2-binding protein TOPBP1 to load replication protein A (RPA) onto the chromatin. Presence of RPA is required for activation and control of replication checkpoints and to undergo repair by homologous recombination. 8 , 9 In the case of management of DNA damage responses like interstrand crosslinks (ICLs), the helicase activity of BACH1 and its interaction with the mismatch repair protein MLH1 provides ICL resistance. 10 BACH1 also acts as a tumor suppressor in different cancer types. 7 , 11 It maintains chromosomal integrity and prevents genomic instability by resolving the G-quadruplexes and processing replication intermediates. 12-14 It has the ability to recognize Gquadruplexes mostly those formed upon replication and mediates their stepwise unfolding and refolding to modulate epigenetic programming and chromatin remodeling also. 12-15 BACH1 maintains as well as preserves the chromatin structure and its epigenetic information hence facilitating the smooth progression of the replication fork when it encounters altered/ damaged/ complex DNA structures ( Figure 2 ). 11 , 12 , 14 As it is involved in regulating many vital pathways, any aberration to it can cause multifactorial diseases like cancer. BACH1/BRIP1 plays a role in hereditary breast and ovarian cancer suppression as well as instrumental in progressive bone marrow failure disorder, Fanconi anemia (FA). 17 Germline mutations in the BACH1/FANCJ gene leads to chromosomal instability which results in bone marrow failure defects, developmental abnormalities and sets up favorable conditions to develop cancer. 18 Clinical data analysis of BRIP1 mutations by Seal et al. indicates that majority of the BRIP1 missense mutants/variants are not linked with a risk of familial breast cancer, whereas the truncated variant of BACH1 are more susceptible alleles of breast cancer running in the family. 19 The biological explanations for the differences in cancer risk for mutant variant and truncated variant are unclear. Moreover, the group identified that, biallelic BRIP1 mutations confers less risk of breast cancer compared to the monoallelic truncated version. 19 Mutations in BACH1 also lead to liver carcinogenesis, among patients with viral cirrhosis, due to impaired DNA mismatch repair pathway. 20 In summary to the above lines, the mutated version of BACH1 gene leads to the development of oncogenicity ( Figure 1 ). BACH1 functionally and physically interact with a bunch of proteins, like BRCA1, MUTLα, MLH1, PMS2, MMS19, TOPBP1, TLS polymerase, BLM, RPA1, MRE11 and FANCD2 and plays a significant role in regulating the metabolic pathways in combination with them. 10 , 21 , 22 The BACH1 interactors play major and minor roles in maintenance of genomic integrity, cell cycle regulation, DNA damage detection and repair processes. 11 The interactors MUTLα, MLH1, PMS2 and RPA1 play important role in mismatch repair. 21 , 23 , 24 The BACH1 homolog, BRIP1 interacts with the mismatch repair heterodimer complex, MUTLα, which is composed of mismatch repair proteins MLH1 and PMS2. It also interacts with MLH1 directly independent of BRCA1. The interaction with the single-stranded DNA-binding protein RPA (Replication Protein A) through its helicase domain enhances the DNA unwinding activities at the difficult sites of replication. 25 The other interactors MMS19 and TOPBP1 maintains genomic integrity with FANCD2 in Fanconi anemia DNA damage repair pathway. 24 The interaction of DNA helicase BACH1 with BLM, another helicase, coordinated with DNA damage signaling protein molecules, structure-specific nucleases, polymerases, RPA, and RAD51 imparts a delicate balance between homologous recombination (HR) and non-homologous end-joining (NHEJ) to repair double strand breaks (DSBs) and maintain genomic stability. 26 The most important event is the physical interaction of BACH1 with BRCA1 which justifies the possible role of BACH1 in cancer development. 7 , 11 This interaction is dependent on the phosphorylation-dephosphorylation function of BACH1, as the interaction increases in presence of phosphatase inhibitors, whereas in the presence of λ phosphatase the interaction is lost, 27 which proves that the phosphorylated form of BACH1 interacts with BRCA1. BRCA1 is localized to the site of DNA double strand break by forming a complex with different interacting molecules like RAP80, CTIP and FANCJ. The BRCA1-RAP80 complex comes through Abraxas ubiquitinase and follows the non-homologous end-joining at the DSBs. The parallel pathway of homologous recombination repair is followed by the BRCA1–FANCJCTIP complex. This complex is also regulated by heterochromatin binding protein 1 (HP1) pathway in response to DNA damage for its accumulation at the site of DNA double strand break which mediates DNA repair. FANCJ interacts with HP1 in a BARD1 dependent manner and mediates homologous recombination. 28 The association of BRCA1 with BACH1 adds on to the functioning of the G2/M checkpoint. 29 The BRCA/BACH1 complex prevents DNA breakage resulting in lowering of genomic instability. 30 BACH1 status affect the recruitment of BRCA1 to double strand breaks depending on the type of damage. 31 Presence of change in amino acid sequence in the BRCA1 binding domain of the protein BACH1, in case of tumorigenesis, proves that recognition of BRCT phosphoprotein by BACH1 is necessary for tumor suppression activity of BRCA1. 32 Schematic representation of BACH1 gene with conserved domains and reported pathogenic mutations in different cancer types. The BACH1 gene comprises of 20 exons of which exon 3&4 belongs to DNA polymerase domain (18-61aa residue), exon 5 nuclear localization signal (NLS; 158-175aa), exon7-19 HBB domain (245-881aa), exon9 DEAH box (393-396aa) and exon 19 &20 DNA helicase/ BRCA1 interacting domain (888-1063aa) respectively. The large boxes represent exons and small colored thick lines represent verified pathogenic mutations in the respective exons in different cancers (data analyzed from cBioPortal.org).