Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ\u27s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases

Structural and Biochemical Analysis of Translesion Synthesis DNA Polymerases

The integrity of DNA is essential for the survival of all living organisms. However, cellular DNA is under constant attack by a variety of internal and external sources. Therefore, cells develop a variety of mechanisms to repair resulting damaged DNA, but some of these lesions remain and are left to encounter the replication machinery. In addition to having the ability to alter the DNA’s coding potential, DNA lesions present severe blocks to normal DNA replication. How these lesions are replicated has been a key question in the areas of DNA replication, mutagenesis, and carcinogenesis. The answer to this long-standing puzzle has come recently with the discovery of a large group of translesion synthesis DNA polymerases, the Y-superfamily. In this work, I will use a variety of biochemical techniques to examine the molecular mechanisms by which these special DNA polymerases are able to bypass DNA that has been damaged by carcinogens

Lymphoma Presenting as Severe Left Ventricular Systolic Dysfunction: A Case Report

Lymphoma involving the heart is rare. This is a case report on non-Hodgkin lymphoma where the patient presented for the first time with heart failure and severe left ventricular systolic dysfunction due to lymphoma infiltrating the heart muscle and had simultaneous bilateral involvement of kidneys. This type of presentation has never been described in world literature and is the first reported case

Role of Human DNA Polymerase k in Extension Opposite from a cis-syn Thymine Dimer

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis–syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3′T of a cis–syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3′T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5′T of the cis–syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3′T of a cis–syn T-T dimer but is remarkably well adapted to accommodate the 5′T via Watson–Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo

Polκ/8-oxoG/dATP ternary complex.

<p>(A) Ribbon diagram representing the overall structure of the ternary complex. The palm, fingers, thumb, PAD and N-clasp domains are shown in cyan, yellow, orange, green, and blue, respectively. DNA is shown in gray and the 8-oxoG lesion and incoming dATP are shown in red. A putative Mg²⁺ ion is shown as a yellow sphere. (B) A view of the ternary complex looking down the DNA helix to show encirclement of the adducted DNA by the N-clasp. (C) Simulated annealing Fo-Fc omit map (contoured at 3.5σ) of 8-oxoG and incoming dATP.</p

Hoogsteen base pairing between 8-oxoG(<i>syn</i>) and incoming dATP.

<p>(A) Close-up view of the Polκ active site cleft. Highlighted and labeled are the catalytic residues (D107, D198, and E199), residues apposed close to incoming dATP (Y112, T138, R144, Y141, and K328), 8-oxoG (M135 and A151), and the base 5<i>′</i> to 8-oxoG (F49 and P153). A putative Mg²⁺ ion is shown as a yellow sphere. (B) Molecular surface representation of the Polκ active site cleft. Highlighted in gray and labeled are residues apposed close to 8-oxoG and the base 5′ to it. A putative Mg²⁺ ion is shown as a yellow sphere.</p

Comparison between Polκ and Dpo4 active site regions.

<p>Close up views of the Polκ (A) and Dpo4 (B) active site regions with template 8-oxoG. Highlighted and labeled are some of the residues implicated in stabilizing 8-oxoG (<i>syn</i>) in the Polκ active site region (M135 and A151) and 8-oxoG (<i>anti</i>) in the Dpo4 active site region (R331, R332 and S34). In the Polκ active site the putative Mg2⁺ ion is shown as a yellow sphere. In the Dpo4 complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005766#pone.0005766-Rechkoblit1" target="_blank">[26]</a>, a water mediated hydrogen bond between R332 and O8 of 8-oxoG in Dpo4 is highlighted. The blue and orange spheres represent the water molecule and the calcium ions respectively. (C) A Hoogsteen 8-oxoG(<i>syn</i>):dATP (<i>anti</i>) base pair in the Polκ structure. (D) A W-C 8-oxoG(<i>anti</i>):dCTP (<i>anti</i>) base pair in the Dpo4 structure.</p