scholarly journals Enzymatic Switching Between Archaeal DNA Polymerases Facilitates Abasic Site Bypass

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.

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

2005 ◽  
Vol 25 (3) ◽  
pp. 1183-1190 ◽  
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.

scholarly journals In VitroGap-directed Translesion DNA Synthesis of an Abasic Site Involving Human DNA Polymerases ϵ, λ, and β

2011 ◽  
Vol 286 (37) ◽  
pp. 32094-32104 ◽  
Author(s):  
Giuseppe Villani ◽  
Ulrich Hubscher ◽  
Nadege Gironis ◽  
Sinikka Parkkinen ◽  
Helmut Pospiech ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Abasic Site ◽  
Translesion Dna Synthesis ◽  
Human Dna ◽  
Translesion Dna

Human DNA Polymerases λ and β Show Different Efficiencies of Translesion DNA Synthesis past Abasic Sites and Alternative Mechanisms for Frameshift Generation†

Biochemistry ◽  
2004 ◽  
Vol 43 (36) ◽  
pp. 11605-11615 ◽  
Author(s):  
Giuseppina Blanca ◽  
Giuseppe Villani ◽  
Igor Shevelev ◽  
Kristijan Ramadan ◽  
Silvio Spadari ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Translesion Dna Synthesis ◽  
Abasic Sites ◽  
Human Dna ◽  
Translesion Dna

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

2002 ◽  
Vol 22 (3) ◽  
pp. 784-791 ◽  
Author(s):  
Lajos Haracska ◽  
Ildiko Unk ◽  
Robert E. Johnson ◽  
Barbara B. Phillips ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Protein A ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Abasic Site ◽  
Nucleotide Incorporation ◽  
Synthesis Activity ◽  
Factor C

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.

scholarly journals Archaeal replicative primases can perform translesion DNA synthesis

2015 ◽  
Vol 112 (7) ◽  
pp. E633-E638 ◽  
Author(s):  
Stanislaw K. Jozwiakowski ◽  
Farimah Borazjani Gholami ◽  
Aidan J. Doherty
Keyword(s):  
Dna Synthesis ◽  
Uv Light ◽  
Small Subunit ◽  
Dna Lesions ◽  
Cyclobutane Pyrimidine Dimers ◽  
Translesion Dna Synthesis ◽  
Template Strand ◽  
Pyrimidine Dimers ◽  
Translesion Dna ◽  
Y Family

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

scholarly journals Differential Roles for DNA Polymerases Eta, Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

2009 ◽  
Vol 30 (5) ◽  
pp. 1217-1230 ◽  
Author(s):  
J. Kevin Hicks ◽  
Colleen L. Chute ◽  
Michelle T. Paulsen ◽  
Ryan L. Ragland ◽  
Niall G. Howlett ◽  
...  
Keyword(s):  
Dna Polymerases ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
Lesion Bypass ◽  
Translesion Dna Synthesis ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Translesion Dna

ABSTRACT Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polη, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.

scholarly journals Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

2007 ◽  
Vol 402 (2) ◽  
pp. 321-329 ◽  
Author(s):  
Giuseppina Blanca ◽  
Emmanuelle Delagoutte ◽  
Nicolas Tanguy le gac ◽  
Neil P. Johnson ◽  
Giuseppe Baldacci ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Dna Polymerases ◽  
Accessory Proteins ◽  
Clamp Loader ◽  
Abasic Site ◽  
Translesion Dna Synthesis ◽  
Translesion Dna

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

scholarly journals Architecture of Y-Family DNA Polymerases Relevant to Translesion DNA Synthesis as Revealed in Structural and Molecular Modeling Studies

2010 ◽  
Vol 2010 ◽  
pp. 1-20 ◽  
Author(s):  
Sushil Chandani ◽  
Christopher Jacobs ◽  
Edward L. Loechler
Keyword(s):  
Conformational Changes ◽  
Dna Polymerases ◽  
Class Versus ◽  
Lesion Bypass ◽  
Translesion Dna Synthesis ◽  
Protein Conformational Changes ◽  
Modeling Studies ◽  
Hydrophobic Amino Acids ◽  
Molecular Modeling Studies ◽  
Y Family

DNA adducts, which block replicative DNA polymerases (DNAPs), are often bypassed by lesion-bypass DNAPs, which are mostly in the Y-Family. Y-Family DNAPs can do non-mutagenic or mutagenic dNTP insertion, and understanding this difference is important, because mutations transform normal into tumorigenic cells. Y-Family DNAP architecture that dictates mechanism, as revealed in structural and modeling studies, is considered. Steps from adduct blockage of replicative DNAPs, to bypass by a lesion-bypass DNAP, to resumption of synthesis by a replicative DNAP are described. Catalytic steps and protein conformational changes are considered. One adduct is analyzed in greater detail: the major benzo[a]pyrene adduct , which is bypassed non-mutagenically (dCTP insertion) by Y-family DNAPs in the IV/-class and mutagenically (dATP insertion) by V/-class Y-Family DNAPs. Important architectural differences between IV/-class versus V/-class DNAPs are discussed, including insights gained by analyzing ~400 sequences each for bacterial DNAPs IV and V, along with sequences from eukaryotic DNAPs kappa, eta and iota. The little finger domains of Y-Family DNAPs do not show sequence conservation; however, their structures are remarkably similar due to the presence of a core of hydrophobic amino acids, whose exact identity is less important than the hydrophobic amino acid spacing.

scholarly journals Non-bulky Lesions in Human DNA: The Ways of Formation, Repair, and Replication

Acta Naturae ◽  
2017 ◽  
Vol 9 (3) ◽  
pp. 12-26 ◽  
Author(s):  
А. V. Ignatov ◽  
K. A. Bondarenko ◽  
A. V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
High Efficiency ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Translesion Dna Synthesis ◽  
Human Dna ◽  
Mechanisms Of Formation ◽  
Bulky Dna Lesions

DNA damage is a major cause of replication interruption, mutations, and cell death. DNA damage is removed by several types of repair processes. The involvement of specialized DNA polymerases in replication provides an important mechanism that helps tolerate persistent DNA damage. Specialized DNA polymerases incorporate nucleotides opposite lesions with high efficiency but demonstrate low accuracy of DNA synthesis. In this review, we summarize the types and mechanisms of formation and repair of non-bulky DNA lesions, and we provide an overview of the role of specialized DNA polymerases in translesion DNA synthesis.

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