Role of error-prone DNA polymerases in spontaneous mutagenesis in Caulobacter crescentus

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

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Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

Journal of Bacteriology ◽

10.1128/jb.186.23.8000-8009.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 8000-8009 ◽

Cited By ~ 26

Author(s):

Michael C. Toporowski ◽

John F. Nomellini ◽

Peter Awram ◽

John Smit

Keyword(s):

Outer Membrane ◽

Caulobacter Crescentus ◽

Outer Membrane Proteins ◽

Functional System ◽

Type I ◽

Wild Type ◽

Residual Level ◽

The Individual ◽

Multicopy Vector

ABSTRACT Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF a and rsaF b were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF a gene is located several kilobases downstream of the other transporter genes, while rsaF b is completely unlinked. An rsaF a knockout had ∼56% secretion compared to wild-type levels, while the rsaF b knockout reduced secretion levels to ∼79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of ∼9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaFa (with normal rsaF b levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

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Role of Y-family translesion DNA polymerases in replication stress: Implications for new cancer therapeutic targets

DNA Repair ◽

10.1016/j.dnarep.2019.03.016 ◽

2019 ◽

Vol 78 ◽

pp. 20-26 ◽

Cited By ~ 9

Author(s):

Peter Tonzi ◽

Tony T. Huang

Keyword(s):

Dna Polymerases ◽

Therapeutic Targets ◽

Replication Stress ◽

Translesion Dna ◽

Y Family

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Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase ζ

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6900-6906.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6900-6906 ◽

Cited By ~ 66

Author(s):

M. Todd Washington ◽

Irina G. Minko ◽

Robert E. Johnson ◽

Lajos Haracska ◽

Thomas M. Harris ◽

...

Keyword(s):

Dna Polymerase ◽

Close Contact ◽

Dna Polymerases ◽

Minor Groove ◽

Synthetic Activity ◽

Oxidation Reactions ◽

Lesion Bypass ◽

Dna Minor Groove

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N 2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

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