Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

ABSTRACT Humans have three DNA polymerases, Polη, Polκ, and Polι, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polκ (hPolκ) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolκ. The processivity of hPolκ, however, is not significantly increased in the presence of these protein factors. The efficiency (V max/K m ) of correct nucleotide incorporation by hPolκ is enhanced ∼50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K m for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased ∼40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolκ would bypass an abasic site by inserting a nucleotide opposite the site.

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Physical and Functional Interactions of Human DNA Polymerase η with PCNA

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7199-7206.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7199-7206 ◽

Cited By ~ 178

Author(s):

Lajos Haracska ◽

Robert E. Johnson ◽

Ildiko Unk ◽

Barbara Phillips ◽

Jerard Hurwitz ◽

...

Keyword(s):

Dna Polymerase ◽

Protein A ◽

Dna Lesions ◽

Nuclear Antigen ◽

Synthetic Activity ◽

Binding Motif ◽

Variant Form ◽

Physical Interaction ◽

Human Dna ◽

Factor C

ABSTRACT Human DNA polymerase η (hPolη) functions in the error-free replication of UV-damaged DNA, and mutations in hPolη cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPolη is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPolη with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPolη inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPolη, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparentK m for the incoming nucleotide.

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A Single Domain in Human DNA Polymerase ι Mediates Interaction with PCNA: Implications for Translesion DNA Synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.1183-1190.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 1183-1190 ◽

Cited By ~ 39

Author(s):

Lajos Haracska ◽

Narottam Acharya ◽

Ildiko Unk ◽

Robert E. Johnson ◽

Jerard Hurwitz ◽

...

Keyword(s):

Dna Synthesis ◽

Tight Binding ◽

Dna Lesions ◽

Nuclear Antigen ◽

Binding Motifs ◽

Translesion Dna Synthesis ◽

Nucleotide Incorporation ◽

Y Family ◽

Stalled Replication Forks ◽

Proliferating Cell

ABSTRACT DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, η, ι, κ, and Rev1, of which Pols η, ι, and κ have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Polδ, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent Km for the nucleotide. Here we show that Polι interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Polι is quite unlike that in Polδ, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.

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Enzymatic Switching Between Archaeal DNA Polymerases Facilitates Abasic Site Bypass

Frontiers in Microbiology ◽

10.3389/fmicb.2021.802670 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xu Feng ◽

Baochang Zhang ◽

Ruyi Xu ◽

Zhe Gao ◽

Xiaotong Liu ◽

...

Keyword(s):

Dna Synthesis ◽

Enzyme Kinetics ◽

Dna Polymerases ◽

Dna Lesions ◽

Abasic Site ◽

Lesion Bypass ◽

Translesion Dna Synthesis ◽

Kinetics Studies ◽

Abasic Sites ◽

Y Family

Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Sulfolobuss islandicus Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4’s activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5–6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an “extender” to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.

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Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases inArchaea

Journal of Bacteriology ◽

10.1128/jb.181.21.6591-6599.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6591-6599 ◽

Cited By ~ 70

Author(s):

Isaac K. O. Cann ◽

Sonoko Ishino ◽

Ikuko Hayashi ◽

Kayoko Komori ◽

Hiroyuki Toh ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Proliferating Cell Nuclear Antigen ◽

Dna Polymerases ◽

Nuclear Antigen ◽

Pol Ii ◽

Circular Dna ◽

Pol I ◽

Factor C ◽

Proliferating Cell

ABSTRACT Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domainEucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (α-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domainsBacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.

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Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Molecular and Cellular Biology ◽

10.1128/mcb.23.14.5107-5112.2003 ◽

2003 ◽

Vol 23 (14) ◽

pp. 5107-5112 ◽

Cited By ~ 55

Author(s):

M. Todd Washington ◽

Sandra A. Helquist ◽

Eric T. Kool ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Hydrogen Bonding ◽

Hydrogen Bonds ◽

Dna Synthesis ◽

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Dna Lesions ◽

Geometric Constraints ◽

High Fidelity ◽

Nucleotide Incorporation

ABSTRACT Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase η (Polη) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Polη are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Polη; in this regard, Polη differs strikingly from classical high-fidelity DNA polymerases.

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Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase ϵ in the presence of replication protein A and proliferating-cell nuclear antigen

Biochemical Journal ◽

10.1042/bj20100405 ◽

2010 ◽

Vol 429 (3) ◽

pp. 573-582 ◽

Cited By ~ 10

Author(s):

Giada A. Locatelli ◽

Helmut Pospiech ◽

Nicolas Tanguy Le Gac ◽

Barbara van Loon ◽

Ulrich Hubscher ◽

...

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Protein A ◽

Replication Protein A ◽

Dna Lesions ◽

Replication Protein ◽

Nuclear Antigen ◽

Abasic Site ◽

Human Dna ◽

Proliferating Cell

DNA pol (polymerase) ϵ is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol ϵ can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol ϵ. RPA inhibited the elongation by human DNA pol ϵ on templates annealed to short primers. PCNA did not influence the elongation by DNA pol ϵ and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol ϵ after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol ϵ to replicate past the AP site, independent of the primer length.

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A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

10.1101/2020.05.18.101550 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kazutoshi Kasho ◽

Gorazd Stojkovič ◽

Cristina Velázquez-Ruiz ◽

Maria Isabel Martínez-Jiménez ◽

Timothée Laurent ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerases ◽

Polymerase Activity ◽

Dna Lesions ◽

Replication Forks ◽

Flexible Loop ◽

Nucleotide Incorporation ◽

Dntp Binding ◽

Incorporation Efficiency

ABSTRACTReplication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Polλ, Polη and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol’s enhanced processivity. PolDIP2 increases PrimPol’s primer-template and dNTP binding affinity, which concomitantly enhances PrimPol’s nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.

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Nontranscriptional Function of FOXO1/DAF-16 Contributes to Translesion DNA Synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.00265-16 ◽

2016 ◽

Vol 36 (21) ◽

pp. 2755-2766 ◽

Cited By ~ 2

Author(s):

Hiroaki Daitoku ◽

Yuta Kaneko ◽

Kenji Yoshimochi ◽

Kaori Matsumoto ◽

Sho Araoi ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Uv Irradiation ◽

Protein A ◽

Dna Lesions ◽

Nuclear Antigen ◽

Transactivation Domain ◽

Content Type ◽

Translesion Dna Synthesis ◽

Translesion Dna

Forkhead box O (FOXO; DAF-16 in nematodes) transcription factors activate a program of genes that control stress resistance, metabolism, and life span. Given the adverse impact of the stochastic DNA damage on organismal development and aging, we examined the role of FOXO/DAF-16 in UV-induced DNA damage response. Knockdown of FOXO1 but not of FOXO3a increases sensitivity to UV irradiation when exposed during S phase, suggesting a contribution of FOXO1 to translesion DNA synthesis (TLS), a replicative bypass of UV-induced DNA lesions. Actually, FOXO1 depletion results in sustained activation of ATR-Chk1 signaling and a reduction of proliferating cell nuclear antigen (PCNA) monoubiquitination following UV irradiation. FOXO1 does not alter the expression of TLS-related genes, but it binds to replication protein A 1 (RPA1), which coats single-stranded DNA and acts as a scaffold for TLS. InCaenorhabditis elegans,daf-16-null mutants show UV-induced retardation in larval development and are rescued by overexpressing a DAF-16 mutant lacking the transactivation domain but not a mutant whose amino acid substitutions render it unable to interact with RPA1. Thus, our findings demonstrate that FOXO1/DAF-16 is a functional component in TLS independent of its transactivation activity.

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