Petr Cejka

DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3′-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5′-terminated strand of the DNA break and to inhibit 3′ to 5′ degradation by Dna2, actions that generate and protect the 3′-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11–Rad50–Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3–Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3–Rmi1 and MRX are important for recruitment of the Sgs1–Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes.

Pif1 family helicases suppress genome instability at G-quadruplex motifs

The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. Here we show that exceptionally potent G-quadruplex unwinding is conserved among Pif1 helicases. Moreover, Pif1 helicases from organisms separated by more than 3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to new genetic and epigenetic changes. Furthermore, when expressed in yeast, human PIF1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.

Methylation‐induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1

The mismatch repair (MMR) gene hMLH1 is mutated in ∼50% of hereditary non‐polyposis colon cancers and transcriptionally silenced in ∼25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR‐proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR‐proficient cells, activation of the MNNG‐induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks

To repair double-strand DNA breaks by homologous recombination, the 5′-terminated DNA strand must first be resected, which generates 3′ single-stranded DNA overhangs. Genetic evidence suggests that this process is initiated by the Mre11–Rad50–Xrs2 (MRX) complex. However, its involvement was puzzling, as the complex possesses exonuclease activity with the opposite (3′ to 5′) polarity from that required for homologous recombination. Consequently, a bidirectional model has been proposed whereby dsDNA is first incised endonucleolytically and MRX then proceeds back to the dsDNA end using its 3′ to 5′ exonuclease. The endonuclease creates entry sites for Sgs1–Dna2 and/or Exo1, which then carry out long-range resection in the 5′ to 3′ direction. However, the identity of the endonuclease remained unclear. Using purified Saccharomyces cerevisiae proteins, we show that Sae2 promotes dsDNA-specific endonuclease activity by the Mre11 subunit within the MRX complex. The endonuclease preferentially cleaves the 5′-terminated dsDNA strand, which explains the polarity paradox. The dsDNA end clipping is strongly stimulated by protein blocks at the DNA end, and requires the ATPase activity of Rad50 and physical interactions between MRX and Sae2. Our results suggest that MRX initiates dsDNA break processing by dsDNA endonuclease rather than exonuclease activity, and that Sae2 is the key regulator of this process. These findings demonstrate a probable mechanism for the initiation of dsDNA break processing in both vegetative and meiotic cells.

Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1–Top3

A double Holliday junction (dHJ) is a central intermediate of homologous recombination that can be processed to yield crossover or non-crossover recombination products. To preserve genomic integrity, cells possess mechanisms to avoid crossing over. We show that Saccharomyces cerevisiae Sgs1 and Top3 proteins are sufficient to migrate and disentangle a dHJ to produce exclusively non-crossover recombination products, in a reaction termed “dissolution.” We show that Rmi1 stimulates dHJ dissolution at low Sgs1–Top3 protein concentrations, although it has no effect on the initial rate of Holliday junction (HJ) migration. Rmi1 serves to stimulate DNA decatenation, removing the last linkages between the repaired and template DNA molecules. Dissolution of a dHJ is a highly efficient and concerted alternative to nucleolytic resolution that prevents crossing over of chromosomes during recombinational DNA repair in mitotic cells and thereby contributes to genomic integrity.

DNA2 drives processing and restart of reversed replication forks in human cells

Following prolonged genotoxic stress, DNA2 and WRN functionally interact to degrade reversed replication forks and promote replication restart, thereby preventing aberrant processing of unresolved replication intermediates

Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic Mismatch Repair

Summary To improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand.

Dependence of the Cytotoxicity of DNA-Damaging Agents on the Mismatch Repair Status of Human Cells

Mismatch repair (MMR) deficiency was reported to increase resistance of mammalian cells to killing by several genotoxic substances. However, although MMR-deficient cells are ∼100-fold more resistant to killing by SN1 type methylating agents than MMR-proficient controls, the sensitivity differences reported for the other agents were typically <2-fold. To test whether these differences were linked to factors other than MMR status, we studied the cytotoxicities of mitomycin C, chloroethylcyclohexyl nitrosourea, melphalan, psoralen-UVA, etoposide, camptothecin, ionizing radiation, and cis-dichlorodiaminoplatinum (cisplatin) in a strictly isogenic system. We now report that MMR deficiency reproducibly desensitized cells solely to cisplatin.

Expression of the MutL Homologue hMLH3 in Human Cells and its Role in DNA Mismatch Repair

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.

The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions.

The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5′- and 3′-single-stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.