Journal article
Visualizing Rev1 catalyze protein-template DNA synthesis
Proceedings of the National Academy of Sciences - PNAS, Vol.117(41), pp.25494-25504
10/13/2020
DOI: 10.1073/pnas.2010484117
PMCID: PMC7568310
PMID: 32999062
Abstract
During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono phosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.
Details
- Title: Subtitle
- Visualizing Rev1 catalyze protein-template DNA synthesis
- Creators
- Tyler M. Weaver - University of Kansas Medical CenterLuis M. Cortez - University of Kansas Medical CenterThu H. Khoang - University of Kansas Medical CenterM. Todd Washington - University of IowaPratul K. Agarwal - Oklahoma State UniversityBret D. Freudenthal - University of Kansas Medical Center
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.117(41), pp.25494-25504
- DOI
- 10.1073/pnas.2010484117
- PMID
- 32999062
- PMCID
- PMC7568310
- NLM abbreviation
- Proc Natl Acad Sci U S A
- ISSN
- 0027-8424
- eISSN
- 1091-6490
- Publisher
- National Academy of Sciences
- Number of pages
- 11
- Grant note
- MCB180199; MCB190044 / National Science Foundation; National Science Foundation (NSF) DEAC0205CH11231 / Department of Energy Office of Science; United States Department of Energy (DOE) R35-GM128562; R01-GM081433; R01-GM105978 / National Institute of General Medical Science; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS)
- Language
- English
- Date published
- 10/13/2020
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984293080502771
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