A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis

Dong, Feng; Weitzel, Steven E.; Hippel, Peter H. von

doi:10.1073/pnas.93.25.14456

Cited by 48 publications

(43 citation statements)

References 29 publications

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“…Helicase rate and processivity are stimulated by coupled polymerase activity (Dong et al 1996;Kim et al 1996;Yuzhakov et al 1996;Stano et al 2005;Hamdan et al 2007). Thus, uncoupling of a blocked leading strand polymerase may enable a limited amount of continued unwinding and lagging strand synthesis, which facilitates restart, while preventing excessive formation of unreplicated gaps in the DNA.…”

Section: Rad53 Regulates Replication Fork Restart Genes and Developmentmentioning

confidence: 99%

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Szyjka¹,

Aparicio²,

Viggiani³

et al. 2008

Genes Dev.

103

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Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3⌬ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.[Keywords: DNA replication fork; DNA damage; DNA repair; cell cycle checkpoint; BrdU; phosphatase; microarray] Supplemental material is available at http://www.genesdev.org.

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Section: Rad53 Regulates Replication Fork Restart Genes and Developmentmentioning

confidence: 99%

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Szyjka¹,

Aparicio²,

Viggiani³

et al. 2008

Genes Dev.

103

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confidence: 99%

“…The polymerase is in turn attached to the clamp protein through a well described interaction between the C terminus of the polymerase and the subunit interfaces of the trimeric clamp protein (5,6,22). Based on functional evidence, an additional interaction between gp41 helicase and the polymerase has been proposed (23,24).gp59 is a central component of the primosome, making protein-protein contacts with all other primosomal proteins. Protein cross-linking experiments have detected molecular contacts between gp59, gp61, gp41, and gp32 proteins (25)(26)(27).…”

mentioning

confidence: 99%

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

Nelson

Yang

Benkovic

2006

Journal of Biological Chemistry

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The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.The T4 replisome is considered to be a model for the more complex replication systems (1, 2). The eight protein replisome can be divided into three modules, the leading strand holoenzyme, the lagging strand holoenzyme, and the primosome (3). The leading and lagging strand holoenzymes are formed by the polymerase (gp43) 4 and the polymerase clamp (gp45) with the aid of the clamp loader protein (gp44/62). Contained within gp43 polymerase are two distinct active sites. One is the polymerization site that catalyzes the 5Ј to 3Ј incorporation of deoxynucleotides (dNTPs) into the growing primer strand, and the other is the exonuclease site that is responsible for the removal of dNMPs in the 3Ј to 5Ј direction (4). gp45 is bound to the polymerase and encircles the DNA duplex, thereby increasing its processivity during DNA synthesis (5, 6). gp44/62 aids in the assembly of the polymeraseclamp complex by positioning the clamp at the 3Ј-OH of the primertemplate junction and acting as a chaperone for the interaction between gp45 and gp43 (7,8). The primosome is made up of the helicase (gp41), primase (gp61), single-stranded DNA-binding protein (gp32), and the helicase loading protein (gp59) (9). gp41 is a hexameric protein that travels along the ssDNA in a 5Ј to 3Ј manner using the energy of ATP hydrolysis to unwind duplex DNA (10, 11). gp61 is also hexameric and is respo...

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“…Besides the Walker A and B boxes (56), which are conserved in all helicases, residues 809 MGITGS 814 of REP pAL1 might correspond to motif III (ϩXϩTGS, where ϩ is hydrophobic and X is any amino acid) of helicase superfamily 2 (20); however, due to the substantial divergence of motif sequences, it is difficult to predict helicase motifs solely from an amino acid sequence (8). Further downstream in the REP pAL1 sequence, a CXXC(X) [13][14] HXXC motif at positions 1155 to 1175 that is also conserved in the rhodococcal homologs might form a binding site for divalent metal ions.…”

Section: Resultsmentioning

confidence: 99%

“…The RecBCD enzyme, for example, showed a maximum rate of between 1,000 and 1,500 bp/s (4, 12). However, since several replicative DNA helicases have been described as much more effective when complexed with the polymerase or with accessory proteins of the replisome (13,28,55), it is tempting to speculate that the high apparent unwinding rate of REP pAL1 in vitro might be due to the presence of modulating domains on the same polypeptide.…”

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confidence: 99%

A Novel Replicative Enzyme Encoded by the LinearArthrobacterPlasmid pAL1

Kolkenbrock

Naumann²,

Hippler³

et al. 2010

J Bacteriol

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The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which codes for the degradation of 2-methylquinoline. Like other linear replicons of actinomycetes, pAL1 is characterized by short terminal inverted-repeat sequences and terminal proteins (TP pAL1 ) covalently attached to its 5 ends. TP pAL1 , encoded by the pAL1.102 gene, interacts in vivo with the protein encoded by pAL1.101. Bioinformatic analysis of the pAL1.101 protein, which comprises 1,707 amino acids, suggested putative zinc finger and topoisomerase-primase domains and part of a superfamily 2 helicase domain in its N-terminal and central regions, respectively. Sequence motifs characteristic of the polymerization domain of family B DNA polymerases are partially conserved in a C-terminal segment. The purified recombinant protein catalyzed the deoxycytidylation of TP pAL1 in the presence of single-stranded DNA templates comprising the 3-terminal sequence (5-GCAGG-3), which in pAL1 forms the terminal inverted repeat, but also at templates with 5-(G/T)CA(GG/GC/CG)-3 ends. Enzyme assays suggested that the protein exhibits DNA topoisomerase, DNA helicase, and DNA-and protein-primed DNA polymerase activities. The pAL1.101 protein, therefore, may act as a replicase of pAL1.Linear plasmids have been identified in higher plants, fungi, and many bacteria. Most of these linear genetic elements are characterized by terminal inverted-repeat sequences and terminal proteins (TPs) covalently attached to their 5Ј ends. The presence of TPs is a consequence of their mode of DNA replication, which in linear plasmids of plants, yeasts, and fungi is initiated at the termini by using the TP as a primer and proceeds by strand displacement. The DNA polymerases encoded by these linear elements are of the viral B type, related to those of contemporary adenoviruses and Bacillus phages (29). The mechanism of protein-primed DNA replication has been studied in detail, especially for the model of Bacillus subtilis phage 29, which uses a monomeric B-family DNA polymerase for both the TP-primed initiation reaction and DNA elongation, resulting in continuous, full-length replication of both strands of the 29 genome (5,6,7,26,42,43,44). In contrast to the linear plasmids of eukaryotes, linear chromosomes and plasmids of Streptomyces spp. replicate bidirectionally from an internal origin (9). This replication mechanism encounters the problem that discontinuous lagging-strand synthesis from RNA-primed Okazaki fragments leaves recessed 5Ј ends at both telomeres when the distal RNA primers are removed. In the Streptomyces linear replicons, the TP serves as a primer for filling in these single-stranded gaps (2, 58). Both the TP and a telomere-associated protein (Tap), which is presumed to recruit the TP and position it at the telomere, are necessary for the propagation of Streptomyces replicons in their linear forms (3).The TP and the Tap protein are highly conserved among many Streptomyces species. On the other hand, several studies have suggested a conside...

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A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis

Cited by 48 publications

References 29 publications

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

A Novel Replicative Enzyme Encoded by the LinearArthrobacterPlasmid pAL1

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