Biological organisms are constantly required to prevent and/or repair damage to genomic DNA. Covalent modification ofthe genetic material is intimately related to processes that contribute to cellular dysfunction. Several mechanisms have evolved to prevent damage to DNA. Once the damage occurs the cell may respond with an arsenal of repair pathways that physically remove the lesions from the genome. If the modified portions of DNA are not removed prior to cell division then the machinery that copies the genetic material, the replisome, will encounter the damage, which often proves inhibitory to accurate DNA replication events. Central to the replisome is an enzyme called a DNA polymerase. The actions of these enzymes play an important role in determining whether a lesion bypass event is accurate or mutagenic. There are several types of DNA polymerase in every cell across the spectrum of life. Some DNA polymerases have been retained throughout evolution because they are an extremely accurate and focused means of synthesizing Watson-Crick base pairs. These so-called replicative DNA polymerases are often less able to bypass DNA adducts. Other specialized polymerases possess lesion bypass abilities that can aid the replication fork when it encounters damage, but how these two types of DNA polymerases are coordinated in response to DNA damage remains unclear. Structural approaches including x-ray crystallography and hydrogen-deuterium exchange mass spectrometry will be combined with functional kinetic analysis and cellular studies in an effort to determine how specialized DNA polymerases interact with the Werner syndrome protein during bypass of damaged DNA. Werner syndrome is characterized by premature aging and genomic instability. Understanding how the enzymes that copy our genome function when they encounter DNA damage is an important part of understandig why certain chemicals are toxic, how cancer develops, and even relates to why we age. The goals of our research seek to answer questions related to how different types of 'DNA making' enzymes function to maintain the integrity of our genetic material.
The proposal is relevant to our global understanding of genomic maintenance. Genomic instability is thought to be a central feature ofthe normal aging process and during tumor development. A more detailed understanding of these processes can lead to better cancer treatments and possibly help improve the overall well-being of individuals as they age.
|Ketkar, Amit; Voehler, Markus; Mukiza, Tresor et al. (2017) Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA. J Biol Chem 292:3154-3163|
|Eddy, Sarah; Tillman, Magdalena; Maddukuri, Leena et al. (2016) Human Translesion Polymerase ? Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA. Biochemistry 55:5218-29|
|Eddy, Sarah; Maddukuri, Leena; Ketkar, Amit et al. (2015) Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA. Biochemistry 54:3218-30|
|Zhao, Linlin; Pence, Matthew G; Eoff, Robert L et al. (2014) Elucidation of kinetic mechanisms of human translesion DNA polymerase ? using tryptophan mutants. FEBS J 281:4394-410|
|Maddukuri, Leena; Ketkar, Amit; Eddy, Sarah et al. (2014) The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa. Nucleic Acids Res 42:12027-40|
|Eddy, Sarah; Ketkar, Amit; Zafar, Maroof K et al. (2014) Human Rev1 polymerase disrupts G-quadruplex DNA. Nucleic Acids Res 42:3272-85|
|Maddukuri, Leena; Shuck, Sarah C; Eoff, Robert L et al. (2013) Replication, repair, and translesion polymerase bypass of N?-oxopropenyl-2'-deoxyadenosine. Biochemistry 52:8766-76|
|Coggins, Grace E; Maddukuri, Leena; Penthala, Narsima R et al. (2013) N-Aroyl indole thiobarbituric acids as inhibitors of DNA repair and replication stress response polymerases. ACS Chem Biol 8:1722-9|
|Ketkar, Amit; Zafar, Maroof K; Maddukuri, Leena et al. (2013) Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity. Chem Res Toxicol 26:221-32|
|Yamanaka, Kinrin; Dorjsuren, Dorjbal; Eoff, Robert L et al. (2012) A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase ?. PLoS One 7:e45032|
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