The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

Abstract

Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3 to 5 DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2⌬405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2⌬405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5 to 3 exonuclease activity of Fen1. Mph1 binds to flapstructured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2⌬405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.Lagging strand DNA synthesis requires the orchestrated actions of many proteins and can be divided into several distinct enzymatic steps (1-4). First, the polymerase (pol) 2 ␣-primase complex synthesizes RNA-DNA primers on the template DNA that are recognized by replication factor C. This complex loads proliferating cell nuclear antigen onto DNA, which acts as a processivity factor tethering pol ␦ to primer ends (5-7). This series of reactions leads to a polymerase switch in which the pol ␣-primase complex at primer ends is displaced and replaced by pol ␦. Okazaki fragments are then elongated by pol ␦ until they encounter downstream Okazaki fragments (2,(8)(9)(10). Pol ␦ continues to synthesize DNA by displacing the 5Ј termini of downstream Okazaki fragments, which generate 5Ј RNA-DNA flap structures (11). These flaps are then cleaved by structure-specific nucleases that lead to the generation of ligatable nicks, which are sealed by DNA ligase converting the noncontiguous lagging strands to a contiguous DNA chain (12, 13).

Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint During DNA Double-Strand Break Repair in Saccharomyces cerevisiae

“…However, Mph1 suppresses BIR (Luke-Glaser and Luke 2012; Stafa et al 2014) and its overexpression increases gross chromosomal rearrangements, apparently by inhibiting HR (Banerjee et al 2008). Mph1 has also been shown to play a role in processing Okazaki fragments during replication (Kang et al 2009). Consistent with previous studies examining the role of Mph1 in ectopic GC (Prakash et al 2009), deleting Mph1 did not affect the efficiency of break repair (when the donors are not separated by a gap) ( Figure 1B).…”

Human Replication Factor C Stimulates Flap Endonuclease 1

“…These include WRN, BLM, Mgs1, PCNA, and Mph1 (62). The acquisition of FEN1 stimulation activity by these proteins may have conferred evolutionary benefits, because such an ability may permit faster generation and sealing of DNA nicks.…”

Interplay between the Smc5/6 complex and the Mph1 helicase in recombinational repair

“…Because Mph1 is also known to be involved in other processes, including lagging-strand synthesis and genomic rearrangements (25,26), the question arises whether the toxicity of Mph1 in mutants of the Smc5/6 complex is related specifically to its recombination functions. We used two approaches to address this issue.…”

“…In the second approach, we examined whether mutations of key residues of the Mph1 helicase domain can suppress defects of smc6 and mms21 mutants, because Mph1 helicase activity is required for its roles in recombination but not for its other functions (15,21,25,26). To this end, we replaced WT MPH1 with mph1-E210Q or mph1-Q603D, which mutated the conserved glutamic acid in the DEAH motif or the key residue of the helicase motif, respectively (15,17).…”

“…Currently, it is unclear how this process is regulated in vivo. In addition to recombination, Mph1 also has possible roles in lagging-strand synthesis and in chromosomal rearrangement when overproduced (25,26). Key residues for Mph1 helicase activity are required only for recombinational repair and not for its other functions (15,21,25,26).…”