RECQL5 and BLM exhibit divergent functions in cells defective for the Fanconi anemia pathway

Abstract

Fanconi anemia (FA) patients exhibit bone marrow failure, developmental defects and cancer. The FA pathway maintains chromosomal stability in concert with replication fork maintenance and DNA double strand break (DSB) repair pathways including RAD51-mediated homologous recombination (HR). RAD51 is a recombinase that maintains replication forks and repairs DSBs, but also rearranges chromosomes. Two RecQ helicases, RECQL5 and Bloom syndrome mutated (BLM) suppress HR through nonredundant mechanisms. Here we test the impact deletion of RECQL5 and BLM has on mouse embryonic stem (ES) cells deleted for FANCB, a member of the FA core complex. We show that RECQL5, but not BLM, conferred resistance to mitomycin C (MMC, an interstrand crosslinker) and camptothecin (CPT, a type 1 topoisomerase inhibitor) in FANCB-defective cells. RECQL5 suppressed, while BLM caused, breaks and radials in FANCB-deleted cells exposed to CPT or MMC, respectively. RECQL5 protected the nascent replication strand from MRE11-mediated degradation and restarted stressed replication forks in a manner additive to FANCB. By contrast BLM restarted, but did not protect, replication forks in a manner epistatic to FANCB. RECQL5 also lowered RAD51 levels in FANCB-deleted cells at stressed replication sites implicating a rearrangement avoidance mechanism. Thus, RECQL5 and BLM impact FANCB-defective cells differently in response to replication stress with relevance to chemotherapeutic regimes.

BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks

“…Intriguingly, co-depletion of BOD1L with another anti-recombinogenic helicase, RECQL5, failed to restore RAD51 foci formation and actually increased/accelerated fork degradation. This is in line with recent data demonstrating that the combined loss of FA proteins with RECQL5 is additive in terms of fork degradation and that BLM and RECQL5 have divergent functions in the absence of an intact FA pathway (Kim et al, 2015).…”

Repair Pathway Choices and Consequences at the Double-Strand Break

“…While limiting HR, RECQL5 counteracts the inhibitory effect of RAD51 on RAD52, thus promoting synthesis-dependent strand annealing (SDSA), which limits COs [56]. Deletion of RECQL5 in Fanconi anemia (FA) cells increases genomic instability, suggesting that RECQL5 in the HR-deficient context can induce compensatory repair mechanisms crucial for survival [57]. Another RecQ helicase, BLM, similarly inhibits the early recombinogenic step of displacing RAD51 from ssDNA in an ATP-dependent manner [58].…”

Protection or resection: BOD1L as a novel replication fork protection factor

“…[5][6][7][8][9][10][11] It seems that the DNA recombinase RAD51 is crucial for fork protection, and defects in RAD51-mediated fork protection underlie the uncontrolled fork resection observed in the absence of many components of the FA/HR pathway. The most wellcharacterized function of RAD51 is its ability to promote strand exchange during HR, by displacing the single-stranded DNA binding protein RPA to form helical nucleofilaments.…”

“…5,6 In addition to RAD51, the FA proteins FANCA, FANCB, FANCD2, PALB2 (FANCN), BRCA1 (FANCS) and BRCA2 (FANCD1) also suppress genomic instability upon replication fork stalling, by preventing the degradation of nascent DNA. 6,11,14 Since BRCA1, PALB2 and BRCA2 are required to load RAD51 onto ssDNA at stalled replication forks, it is natural to assume that their ability to protect replication forks from degradation is due to their role in this process. However, studies of a BRCA2 mutant that lacks a conserved C-terminal RAD51-binding site revealed that this region is essential for fork protection but dispensable for loading RAD51 onto DNA, and for HR.…”

“…MRE11 has been implicated in the uncontrolled resection observed in the absence of many factors, including a functional FA/HR pathway: inhibition of MRE11 in the absence of FANCA, FANCB, BRCA1, BRCA2, FANCD2, RECQL5, REV1 and PARP1 prevents fork resection. 5,6,9,11,15 Moreover, MRE11-dependent fork resection underlies the increased chromosome breakage exhibited by BRCA2 null cells. 5 Thus it seems that these FA/HR factors specifically restrict the activity of MRE11 at replication forks to prevent over-processing.…”