Structural Basis for FEN-1 Substrate Specificity and PCNA-Mediated Activation in DNA Replication and Repair

Chapados, Brian R.; Hosfield, David J.; Han, Seungil; Qiu, Junzhuan; Yelent, B.; Shen, Binghui; Tainer, John A.

doi:10.1016/s0092-8674(03)01036-5

Cited by 266 publications

(389 citation statements)

References 50 publications

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“…7B nonetheless show that the DNA is bent in the Taq polymerase-DNA complex, forming an angle of about 120°presumably at the junction between the upstream duplex and downstream duplex. Such upstream and downstream duplexes bending was also observed in P. furiousus flap endonuclease I interactions with DNA substrates by molecular modeling (41), and in the crystal structure of Archaeoglobus fulgidus flap endonuclease I in complex with a similar overlap flap DNA substrate (42). DISCUSSION Our SANS results suggest that, when Taq polymerase binds the overlap flap DNA substrate, tethered Klentaq and 5Ј-nuclease domains and their accompanying active sites do not approach closely enough to allow the simultaneous upstream primer extension and downstream primer cleavage, as speculated, to generate a ligatable nick.…”

Section: Resultsmentioning

confidence: 61%

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Byrnes

et al. 2004

Journal of Biological Chemistry

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The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5 -nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNAbound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.In addition to its well known in vitro applications in PCR, in vivo the DNA polymerase I from Thermus aquaticus (Taq polymerase) performs nucleotide replacement reactions in DNA repair and RNA primer removal in Okazaki fragment processing during lagging-strand synthesis (1). Taq polymerase also utilizes both its polymerase and 5Ј-nuclease activities in a nucleotide replacement reaction: it catalyzes the upstream addition of deoxynucleoside 5Ј-triphosphates to the 3Ј-hydroxyl terminus of an RNA primer and also cleaves the downstream, single-stranded 5Ј-nucleotide displaced by the growing upstream strand. On a DNA duplex substrate, upstream primer extension and downstream strand excision must be highly coordinated (2) to leave a ligatable nick, instead of a gap or an overhang, on a DNA duplex that is to be sealed later by a DNA ligase.The structure and functional properties of Taq polymerase are similar to those of the polymerase I of Escherichia coli (3). Taq polymerase has two distinct domains: an N-terminal domain (residues 1-289) that has single-stranded 5Ј-nucleotide cleavage activity (5Ј-nuclease domain), and a C-terminal domain (residues 306 -832) that has DNA polymerase activity (Klentaq domain). Integrated into the Klentaq domain is another domain (residues 306 -423) that is structurally analogous to the proof editing 3Ј 3 5Ј-exonuclease domain of E. coli polymerase I, but this domain has n...

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Section: Resultsmentioning

confidence: 61%

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Byrnes

et al. 2004

Journal of Biological Chemistry

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“…In elegant genetic studies, Wu et al (16) provided evidence that the DNA structure endonuclease FEN-1(Rad27) is involved in nucleolytic processing events that occur after end alignment in NHEJ. The participation of FEN-1(Rad27), a multifunctional enzyme, in DNA replication and the long patch subpathway of base excision repair is mediated by an interaction with proliferating cell nuclear antigen (13,17,(21)(22)(23). In this study, we identified and characterized physical and functional interactions between FEN-1(Rad27) and both Pol4 and Dnl4/Lif1, the enzymes that catalyze the gap-filling and ligation steps of NHEJ, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…FEN-1 is member of the growing family of proteins that bind to the interdomain connector loop of proliferating cell nuclear antigen, which functions as a ring-shaped homotrimer (17)(18)(19)(20). This interaction with proliferating cell nuclear antigen, which involves residues in addition to the conserved proliferating cell nuclear antigen binding motif, stimulates the DNA structurespecific endonuclease activity of FEN-1 and presumably contributes to the participation of FEN-1 in Okazaki fragment maturation and long patch base excision repair (13,17,(21)(22)(23). Although genetic studies in yeast indicate that FEN-1(Rad27) is involved in the processing of aligned NHEJ intermediates with 5Ј flaps (16,24), the protein-protein interactions that specifically recruit FEN-1 to the NHEJ intermediates have not been identified.…”

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confidence: 99%

Processing and Joining of DNA Ends Coordinated by Interactions among Dnl4/Lif1, Pol4, and FEN-1

Tseng

Tomkinson

2004

Journal of Biological Chemistry

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The repair of DNA double-strand breaks is critical for maintaining genetic stability. In the non-homologous end-joining pathway, DNA ends are brought together by end-bridging factors. However, most in vivo DNA double-strand breaks have terminal structures that cannot be directly ligated. Thus, the DNA ends are aligned using short regions of sequence microhomology followed by processing of the aligned DNA ends by DNA polymerases and nucleases to generate ligatable termini. Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structurespecific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. In this study, we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5 ends. Because Pol4 also interacts with Dnl4/Lif1, our results have revealed a series of pair-wise interactions among the factors that complete the repair of DNA doublestrand breaks by non-homologous end-joining and provide a conceptual framework for delineating the endprocessing reactions in higher eukaryotes.The repair of DNA double-strand breaks (DSBs) 1 is critical for the maintenance of genomic integrity and stability. There are two main DSB repair pathways in eukaryotes: homologous recombination and non-homologous end joining (NHEJ) (1). In the error-free homologous recombination pathway, an intact homologous DNA duplex acts as a template for repair, resulting in the accurate restoration of the broken DNA molecule. By contrast, in NHEJ, the two broken ends are simply rejoined to one another in a process that frequently causes loss and/or gain of nucleotides at the break site and occasionally results in the joining of previously unlinked DNA molecules. Notably, defects in the repair of DSBs by either homologous recombination or NHEJ have been linked with cancer predisposition (2, 3).Studies with mammalian cells identified the DNA-dependent protein kinase (DNA-PK), which is composed of the DNA end binding Ku70/Ku80 heterodimer and the DNA-PK catalytic subunit, and the DNA ligase IV/XRCC4 complex as key NHEJ factors (4). In Saccharomyces cerevisiae, Hdf1/Hdf2 and Dnl4/Lif1 are the functional homologs of Ku70/Ku80 and DNA ligase IV/XRCC4, respectively (4). Although yeast lacks a DNA-PK catalytic subunit homolog, genetic studies have revealed that the Rad50/Mre11/Xrs2 complex is a key player in NHEJ (4 -7). Using purified protein complexes, it has been shown that the Rad50/Mre11/Xrs2 complex has robust endbridging activity and specifically stimulates intermolecular DNA joining by Dnl4/Lif1 (8). Furthermore, efficient intermolecular DNA joining mediated by the Rad50/Mre11/Xrs2 and Dnl4/Lif1 complexes was dependent upon Hdf1/Hdf2 at physiological salt concentrations (8). Based on these results, it seems likely that Hdf1/Hdf2 enhances the recruitment of the Rad50/ Mre11/Xrs2 end-bridging complex to DNA ends and that Dnl4/ Lif1 joins the res...

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“…PCNA is a highly conserved eukaryotic homotrimeric protein that assembles around DNA to form a sliding clamp and acts as a processivity factor for the replicative polymerase (reviewed in [16]). PCNA interacts with a large variety of proteins involved in DNA replication, lesion bypass, DNA repair, and cell cycle control, such as DNA polymerases δ and ε [17], DNA polymerase η [18], LIGI [19], MSH3 and MSH6 [20], FEN1 [21], GADD45 [22], and p21 [23]. An eight amino acid conserved motif (Q-x-x-[I/L/M]-x-x-F-[F/ Y]) in these proteins modulates their binding to PCNA [24].…”

Section: Introductionmentioning

confidence: 99%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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Structural Basis for FEN-1 Substrate Specificity and PCNA-Mediated Activation in DNA Replication and Repair

Cited by 266 publications

References 50 publications

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Processing and Joining of DNA Ends Coordinated by Interactions among Dnl4/Lif1, Pol4, and FEN-1

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

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