Mol Bio 3II3 (T05)Research Summary AssignmentJeevika Goyal (1209306)Breast and ovarian cancer type 1 susceptibility protein (BRCA1) is part of tumor suppressing genes in our body. It plays an integral role in double stranded DNA repair pathway, cell cycle check point and binds to various transcription factor to ensure that DNA repair is completed without hindrance. It is expressed in actively proliferating cells and can lead to potential genomic instability. Mutations in this proteins accounts for over 20% of hereditary breast cancers in United stated. Therefore, furthering our understanding of this protein is critical as it may provide potential treatment for breast cancer or provide potential ways to decrease the chances of inheriting breast cancer. Previous studies indicates that BRCA1 plays a role in DNA damage repair during S and G2 cell cycle. However, BRCA1s role in repair of DNA damage from bulky DNA adducts has not been explored. Additionally, it was also observed that many of the BRCA1 interacting proteins also play a role in repair response of interstrand crosslinking (ICL). Therefore, the purpose of this paper is to explore BRCA1s role in the post replication repair triggered by UV damage. This study strives to provide a molecular explanation of BRCA1s role in repair response to stalled replication forks, as they hypothesize that BRCA1s role in this pathway differs from its function in the Double stranded break repair (DSB). Furthering our understanding of BRCA1 may be important To address this question, researchers first confirm the effects of BRCA1 deficiency in human breast cancer cells in response to UV-C damage. To test this, they subjected four BRCA1-/- human break cancer cell lines (HCC1937, SUM1315, L5BRC1 and SUM149) to UV C irradiation. They observed the colony growth after 7 days and observed that there was decrease in these BRCA1-/- cell lines. Additionally, decrease in sensitivity to UV is observed when HCC1937 is reengineered in conjunction with wildtype, in an attempt to restore some of the wildtype response. Next step was to understand the recruitment of BRCA1 to the sites of UV damage. To investigate this U2OS cells were then overlaid on filters where they were UV irradiated and fixed. Damaged locations were detected using antibody to CPD and Immunofluorescence was used to detect the recruitment of BRCA1 to the damage site. Results indicate that during damage induced by UV, recruitment of BRCA1 at damage site was observed as, BRCA1 staining and CPD staining colocalized. Additionally, researchers used aphidicolin to block the replication fork and subjected it to UV irradiation, to examine the levels of BRCA1 during the cell cycle. Controls indicate BRCA1 levels are low in G0 and G1 phase but increase in S phase; however, when the replication is blocked BRCA1 levels dropped significantly, indicating that BRCA1 recruitment is replication dependent. Nucleotide excision repair pathway (NER) function in repairing UV induced lesions in DNA by recognizing photoproducts. However, since BRCA1 was not observed to be involved in the recognition of these photoproducts, researchers hypothesized that its accumulation at the damage site may be dependent upon prior recruitment of NER proteins to damage site. To analyze this, three strains of cells, WT, XPA deficient and XPC deficient were subjected to UV. When cells were observed 1 hours later, greater recruitment of BRCA1 was detected in XPA- deficient and XPC deficient cell lines; thus, indicating that BRCA1 recruitment is NER independent. Therefore, since BRCA1 is replication dependent but NER independent, it may indicate that BRCA1 plays a role in formation of stalled replication forks at sites with residual photoproducts. Researchers further, hypothesized that BRCA1 may then play a role in excision of these photoproducts. To investigate this BRCA1- depleted culture was analyzed at three stages of cell cycle G1, S and G2 using 6-4PP and CPD excision analysis. Decrease in both 6-4PP and CRP excision rate of cells in S phase and some in G phase was observed. This indicates that BRCA1 is required for photoproduct excision; however, S and G2 cells require accumulation of a certain concentration of BRCA1 for it to eliminate photoproduct. Results also indicate that this optimal level of BRCA1 is required for recruitment of ERCC1, an important component of NER mediated photoproduct elimination.Researchers also investigate the interaction between BRCA1 and replication factor C (RFC) complex to further their understanding of BRCA1 response to UV initiated stalled replication forks. To analyze this researchers used antibodies for three RFC subunits (RFC1, RFC4 and RFC5). These antibodies were present near the UV damage site at the stalled replication site in the same areas as the BRCA1; indicating that BRCA1 may be required for stable association between these three RFC subunits at the stalled fork. Additionally, co-depletion of RFC2 also led to co-deletion of BRCA1. This was observed by decrease in BRCA1 binding to damage sites ShRFC2 cells compare to ShLuc (positive control) cells. Thus, the results indicates that BRCA1 mediates the of behaviour RFC complex at the stalled replication fork.Researchers of this paper use appropriate controls for every experiment and used multiple techniques to verify their results. Therefore, I support the conclusion provided by the researchers as they present convincing data from multiple experiments to support their hypothesis about the role of BRCA1 in other DNA repair mechanisms in addition to DNA repair pathway. An experiment that could have been included as part of this study could be actually depicting that ERCC1 recruitment is required for excision of photoproduct. This is because they repeatedly draw inferences that because ERCC1 is recruited post BRCA1 accumulation; therefore, it may be important for the elimination of photoproducts at those locations. However, they do not perform a conclusive experiment to show this. Additionally, I found that the paper included a lot of diagrams. I think that researchers should have included only summarized data, and all the additional data could have been part of the supplementary data, as all of the diagrams were neither referenced nor explained within the paper. Also, authors explain the techniques within the research paper. This is helpful to some extent; however, I would have preferred if they had explained this in the methods section of their paper.

References: Li M.L, Greenberg A.R. Links between genome integrity and BRCA1 tumor suppression. Trends. 37 (10), 418 -424. (2012)

Pathania S, Nguyen J, Hill SJ, Scully R, Adelmant GO, Marto JA, Feunteun J, Livington DM. BRCA1 is required for postreplication repair after UV induced DNA damage. Molecular Cell. 44, 235- 251 (2011).

Rosen E.M. BRCA1 in the DNA damage response and at telomeres. Front Genet. 4, 85. (2013)

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