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).

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

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