Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

Abstract

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G 1 , after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.The RecQ helicases are a family of DNA-unwinding enzymes essential for the maintenance of genome integrity in all kingdoms of life. Five RecQ enzymes have been found in human cells: RECQ1 (also called RECQL or RECQL1), BLM (RECQ2 or RECQL3), WRN (RECQ3 or RECQL2), RECQ4 (RECQL4), and RECQ5 (RECQL5) (3, 7). Here we refer to these helicases as RECQ1, RECQ4, and RECQ5, without the "L" that is present in the official gene names. Mutations in the BLM, WRN, and RECQ4 genes are linked to Bloom syndrome (BS), Werner syndrome (WS), and the subset of RothmundThomson syndrome (RTS) patients at high risk of developing osteosarcomas, respectively (19,31,71). RECQ4 mutations have also been associated with RAPADILINO and BallerGerold syndrome (56, 61). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that BLM, WRN, and RECQ4 are involved in different aspects of DNA metabolism. However, the molecular events underlying the pathogenesis of BS, WS, and RTS remain obscure. Mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5, have not as yet been identified as causes of either genomic instability or heritable cancer predisposition disorders.Several lines of evidence suggest that RecQ helicases play an important role in DNA replication control (3, 10). In particular, RecQ helicases are thought to facilitate replication by preserving the integrity of stalled replication forks and by remodeling or repairing damaged or collapsed forks to allow the resumption of replication. Consistent with these ideas, several investigators have shown that primary fibroblasts from BS, WS, and RTS patients and RecQ5-deficient mouse embryonic fibroblasts all show differential...

RECQ1 is required for cellular resistance to replication stress and catalyzes strand exchange on stalled replication fork structures

“…45 Interestingly, both RECQ1 and WRN are identified as S-phase population, with an effect on the rate of DNA synthesis with lower BrdU incorporation. 10 Consistently, BrdU staining in the RECQ1-depleted cells is relatively lower than in the scrambled cells (Fig. S3A).…”

“…42 Consistently, a defect in the frequency of origin firing was reported after transient depletion of RECQ1 along with a decrease in replication fork progression rate. 10 Phosphorylation of RPA and activation of CHK1 are also considered important consequences for the cellular signaling in response to DNA damage. More interestingly, RECQ1 accumulation of stalled/collapsed replication forks in the absence of RECQ1 would eventually trigger recombination repair and an increase in HR events (Fig.…”

“…11,12 RECQ1 associates with the origins specifically in S-phase and is speculated to play an important role in replication fork elongation, 10 but its functional role in the stabilization and progression of stalled replication forks has not been described.…”

“…Both RECQ1 and RECQ4 were proposed to be associated with DNA replication origins in a cell cycle-dependent manner. 10 More specifically, RECQ4 associates with the origins in the late G 1 phase and is involved in the initiation of DNA replication and loading of replication factors such as RPA, PCNA and polymerase α.…”

Overexpression of RECQL4 is associated with poor prognosis in patients with gastric cancer

“…As a key factor of the DNA unwinding helicase, RECQL4 is involved in cell processes (20). A tumor-promoting function of RECQL4 has been widely described (21).…”