FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells

Abstract

In order to exploit the specific vulnerabilities of tumors, it is urgent to identify the basis of associated defects in genome maintenance. One unsolved problem is the mechanism of inhibition of processing of DNA double-strand break repair by REV7 and its influence on DNA repair pathways. We searched for REV7-associated proteins in human cells and found FAM35A, a protein of previously unknown function. By analyzing the FAM35A sequence we discovered that FAM35A has an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded binding protein RPA. Knockdown of FAM35A caused sensitivity to DNA damaging agents, and FAM35A re-localized in damaged cell nuclei. In a BRCA1 mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. Moreover, we found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations in cancer revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases. The results reveal a new DNA repair factor with promise as a therapeutically relevant cancer marker.

SHLD 2/ FAM 35A co‐operates with REV 7 to coordinate DNA double‐strand break repair pathway choice

“…In a series of functional studies, we show that SHLD2 is critical during both antibody diversification and DSB repair by the NHEJ pathway. Our data suggest that SHLD2 and REV7 act together in an epistatic manner, which is corroborated by several studies that described SHLD2 as a novel DNA repair factor (Barazas et al , ; Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Mirman et al , ; Noordermeer et al , ; Tomida et al , ). We further show that, similar to 53BP1 and RIF1 (Chapman et al , , ; Di Virgilio et al , ; Escribano‐Diaz et al , ; Feng et al , ; Zimmermann et al , ), SHLD2 opposes HR by limiting DNA end resection.…”

“…Here, we provide further insight into the role of SHLD2 in DNA repair and show that SHLD2 acts as a downstream effector of REV7 in the NHEJ pathway. Through its N‐terminal domain, SHLD2 is mobilized to and accumulates at sites of DNA damage in a 53BP1‐, RIF1‐, and REV7‐dependent manner, in accordance with several recent studies describing the role of SHLD2 in the DNA damage response (Barazas et al , ; Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Mirman et al , ; Noordermeer et al , ; Tomida et al , ). Importantly, we show that the N‐terminus of SHLD2 has very limited DNA‐binding capacity, which support a model where the recruitment of SHDL2 to DSBs is promoted by protein–protein interactions (Noordermeer et al , ).…”

“…However, it remains largely unclear how they modulate the proper balance between NHEJ and HR. Several recent studies have recently tackled to decipher the effectors of the 53BP1-RIF1-REV7 axis Dev et al, 2018;Ghezraoui et al, 2018;Gupta et al, 2018;Mirman et al, 2018;Noordermeer et al, 2018;Tomida et al, 2018), and our work contributes to this effort by providing further insight into the role of REV7 in DNA repair pathway choice.…”

“…By performing structure prediction analyses on SHLD2 protein sequence using Motif Scan (MyHits, SIB, Switzerland) and InterProScan5 (Jones et al , ), we identified a putative N‐terminal DNA‐binding domain (NUMOD3 domain) and a structural motif in its C‐terminus that we labeled PFAM (Fig A). Recently, structural prediction analyses of SHLD2 also defined a N‐terminal motif promoting protein–protein as well as three putative OB‐fold like domains at its C‐terminus (Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Noordermeer et al , ; Tomida et al , ). Finally, previous phospho‐proteomic analysis identified a S/Q substrate of ATM/ATR following DNA damage at position 339 (Matsuoka et al , ).…”

“…Recently, structural prediction analyses of SHLD2 also defined a Nterminal motif promoting protein-protein as well as three putative OB-fold like domains at its C-terminus (Dev et al, 2018;Ghezraoui et al, 2018;Gupta et al, 2018;Noordermeer et al, 2018;Tomida et al, 2018). Finally, previous phospho-proteomic analysis identified a S/Q substrate of ATM/ATR following DNA damage at position 339 (Matsuoka et al, 2007).…”