Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation. The association of neutropenia and NK cell deficiency, which is unusual among primary immunodeficiencies and bone marrow failures, was due to a blockade in the bone marrow and was mildly symptomatic. We discovered compound heterozygous rare mutations in Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1, also known as PSF1) in the 5 patients. The GINS complex is essential for eukaryotic DNA replication, and homozygous null mutations of GINS component-encoding genes are embryonic lethal in mice. The patients' fibroblasts displayed impaired GINS complex assembly, basal replication stress, impaired checkpoint signaling, defective cell cycle control, and genomic instability, which was rescued by WT GINS1. The residual levels of GINS1 activity reached 3% to 16% in patients' cells, depending on their GINS1 genotype, and correlated with the severity of growth retardation and the in vitro cellular phenotype. The levels of GINS1 activity did not influence the immunological phenotype, which was uniform. Autosomal recessive, partial GINS1 deficiency impairs DNA replication and underlies intra-uterine (and postnatal) growth retardation, chronic neutropenia, and NK cell deficiency.

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.

Background:The biochemical function of variable N-terminal regions of eukaryotic Dna2 remains unclear. Results: The N-terminal 45-kDa domain of yeast Dna2 targets the enzyme specifically to a secondary structure flap. Conclusion:The hairpin binding activity of Dna2 contributes to efficient removal of hairpin flaps together with endonuclease and helicase activities during Okazaki fragment processing. Significance: The hairpin binding activity of Dna2 is critical for genome stability.

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3′-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Δ405N devoid of the N-terminal 405-amino-acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Δ405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that vts1Δ dna2Δ405N and vts1Δdna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and in vitro evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki fragment processing.