scholarly journals Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis

2019 ◽  
Vol 20 (20) ◽  
pp. 5012 ◽  
Author(s):  
Ondrej Hrabina ◽  
Viktor Brabec ◽  
Olga Novakova
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Duplexes ◽  
Translesion Dna Synthesis ◽  
Processing Enzymes ◽  
Microscale Thermophoresis ◽  
Oxidative Products ◽  
Dna Processing ◽  
Damaged Dna

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2’-deoxyribo-5-hydroxyuridin (HdU) and 2’-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF–), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

scholarly journals Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli

2008 ◽  
Vol 191 (2) ◽  
pp. 665-672 ◽  
Author(s):  
Susan E. Cohen ◽  
Veronica G. Godoy ◽  
Graham C. Walker
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Temperature Sensitivity ◽  
Dna Polymerases ◽  
Rna Polymerases ◽  
Translesion Dna Synthesis ◽  
Catalytic Activities ◽  
Translesion Dna

ABSTRACT NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.

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 The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1083
Author(s):  
Adhirath Sikand ◽  
Malgorzata Jaszczur ◽  
Linda B. Bloom ◽  
Roger Woodgate ◽  
Michael M. Cox ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Pathogenic Bacteria ◽  
Fold Increase ◽  
Translesion Dna Synthesis ◽  
Sliding Clamp ◽  
Pol V ◽  
Dna Polymerase V ◽  
Acting In Concert

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase

Mutagenesis ◽  
2019 ◽  
Author(s):  
Zhenyu Zou ◽  
Tingting Liang ◽  
Zhongyan Xu ◽  
Jiayu Xie ◽  
Shuming Zhang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Accessory Proteins ◽  
Abasic Site ◽  
Traditional Concept ◽  
Translesion Dna Synthesis ◽  
Dna Lesion ◽  
Translesion Dna

Abstract Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme–DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

scholarly journals Mechanism of Translesion DNA Synthesis by DNA Polymerase II

1996 ◽  
Vol 271 (40) ◽  
pp. 24662-24669 ◽  
Author(s):  
Tamar Paz-Elizur ◽  
Masaru Takeshita ◽  
Myron Goodman ◽  
Michael O'Donnell ◽  
Zvi Livneh
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Translesion Dna Synthesis ◽  
Translesion Dna ◽  
Dna Polymerase Ii
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