DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase.

Download Full-text

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

Cells ◽

10.3390/cells10051083 ◽

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.

Download Full-text

Mutant forms of theEscherichia coliβ sliding clamp that distinguish between its roles in replication and DNA polymerase V-dependent translesion DNA synthesis

Molecular Microbiology ◽

10.1111/j.1365-2958.2005.04500.x ◽

2005 ◽

Vol 55 (6) ◽

pp. 1751-1766 ◽

Cited By ~ 33

Author(s):

Mark D. Sutton ◽

Jill M. Duzen ◽

Robert W. Maul

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Translesion Dna Synthesis ◽

Sliding Clamp ◽

Dna Polymerase V ◽

Translesion Dna ◽

Mutant Forms

Download Full-text

Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated

Journal of Bacteriology ◽

10.1128/jb.184.10.2674-2681.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2674-2681 ◽

Cited By ~ 34

Author(s):

Angela Borden ◽

Paul I. O'Grady ◽

Dominique Vandewiele ◽

Antonio R. Fernández de Henestrosa ◽

Christopher W. Lawrence ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Wild Type ◽

Lesion Bypass ◽

Base Pairs ◽

Pol V ◽

Pol I ◽

Cyclobutane Dimer ◽

Dna Polymerase V ◽

Pol Iii

ABSTRACT Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

Download Full-text

Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.20.6953-6961.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 6953-6961 ◽

Cited By ~ 35

Author(s):

Charmain T. Courcelle ◽

Jerilyn J. Belle ◽

Justin Courcelle

Keyword(s):

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Translesion Synthesis ◽

Replication Forks ◽

Repair Capacity ◽

Translesion Dna Synthesis ◽

Pol V ◽

Nucleotide Excision ◽

Translesion Dna

ABSTRACT Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.

Download Full-text

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.172197099 ◽

2002 ◽

Vol 99 (17) ◽

pp. 11061-11066 ◽

Cited By ~ 40

Author(s):

P. Pham ◽

E. M. Seitz ◽

S. Saveliev ◽

X. Shen ◽

R. Woodgate ◽

...

Keyword(s):

Dna Polymerase ◽

Translesion Synthesis ◽

Dna Polymerase V

Download Full-text

Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword

Biochemistry ◽

10.1021/acs.biochem.6b00117 ◽

2016 ◽

Vol 55 (16) ◽

pp. 2309-2318 ◽

Cited By ~ 16

Author(s):

Malgorzata Jaszczur ◽

Jeffrey G. Bertram ◽

Andrew Robinson ◽

Antoine M. van Oijen ◽

Roger Woodgate ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerase V

Download Full-text

Investigating the role of the E. coli β‐sliding clamp in DNA polymerase V‐dependent translesion DNA synthesis

The FASEB Journal ◽

10.1096/fasebj.20.5.a909-c ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

Mark David Sutton ◽

Laurie Sanders ◽

Sarah Ponticelli ◽

Jill Duzen ◽

Robert Maul ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Translesion Dna Synthesis ◽

E Coli ◽

Sliding Clamp ◽

Dna Polymerase V ◽

Translesion Dna

Download Full-text

Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

Nature Communications ◽

10.1038/s41467-021-24317-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Olga Rechkoblit ◽

Robert E. Johnson ◽

Yogesh K. Gupta ◽

Louise Prakash ◽

Satya Prakash ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Thermodynamic Stability ◽

Active Site ◽

Base Pair ◽

Translesion Synthesis ◽

Human Cells ◽

Structural Basis ◽

Human Dna ◽

Dna Template

AbstractPrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

Download Full-text