Biochemical analysis of human Dna2

Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5'–3' nuclease activity with preference for single-stranded 5' flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5' end. Moreover, hDna2 shows strong 3'–5' nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5'–3' activity, is suppressed by steric hindrance at the 3'-end, suggesting that the 3'–5' nuclease requires a 3' single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3'–5' nuclease activity may be more important than previously thought

Single Strand Annealing and ATP-independent Strand Exchange Activities of Yeast and Human DNA2: possible role in Ozaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented

Processing of G4 DNA by Dna2 Helicase/nuclease and RPA provides insights into the mechanism of Dna2/RPA substrate recognition

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening

The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts

BACKGROUND: DNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol α and Pol δ in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol ε in chromosomal DNA replication is not well understood. RESULTS: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol ε. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol ε-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol ε holoenzyme to Pol ε-depleted extracts, but not by addition of polymerase-deficient forms of Pol ε, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol ε holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells. CONCLUSION: These results indicate that the DNA polymerase activity of Pol ε holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol ε holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts

Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae

Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5'-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing

Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5'-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5'-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of ~5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing

Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5′ single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5′ single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn^(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity

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

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway