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Title: Activity and fidelity of human DNA polymerase $α$ depend on primer structure

Abstract

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA–DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure–function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA–DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the “fingers” subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Furthermore, pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end.more » A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [2];  [1]
  1. Univ. Nebraska Medical Center, Omaha NE
  2. Yale Univ. School of Medicine, New Haven, CT (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH), National Institute of General Medical Sciences (NIGMS); Cancer Center Support Grant; USDOE Office of Science (SC); Fred and Pamela Buffett Cancer Center Support Grant
OSTI Identifier:
1439639
Grant/Contract Number:  
GM101167; GM49551; 5T32GM06754 3-12; P30CA036727; P41 GM103403; AC02-06CH11357; INBRE-P20GM103427-14; COBRE-1P30GM110768-01
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 293; Journal Issue: 18; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA replication; DNA polymerase; human; crystal structure; DNA-protein interaction; RNA; conformational change; chimeric RNA-DNA primer; Polα; Polδ; Polε; pre-steady-state kinetics

Citation Formats

Baranovskiy, Andrey G., Duong, Vincent N., Babayeva, Nigar D., Zhang, Yinbo, Pavlov, Youri I., Anderson, Karen S., and Tahirov, Tahir H. Activity and fidelity of human DNA polymerase $α$ depend on primer structure. United States: N. p., 2018. Web. doi:10.1074/jbc.RA117.001074.
Baranovskiy, Andrey G., Duong, Vincent N., Babayeva, Nigar D., Zhang, Yinbo, Pavlov, Youri I., Anderson, Karen S., & Tahirov, Tahir H. Activity and fidelity of human DNA polymerase $α$ depend on primer structure. United States. doi:10.1074/jbc.RA117.001074.
Baranovskiy, Andrey G., Duong, Vincent N., Babayeva, Nigar D., Zhang, Yinbo, Pavlov, Youri I., Anderson, Karen S., and Tahirov, Tahir H. Mon . "Activity and fidelity of human DNA polymerase $α$ depend on primer structure". United States. doi:10.1074/jbc.RA117.001074. https://www.osti.gov/servlets/purl/1439639.
@article{osti_1439639,
title = {Activity and fidelity of human DNA polymerase $α$ depend on primer structure},
author = {Baranovskiy, Andrey G. and Duong, Vincent N. and Babayeva, Nigar D. and Zhang, Yinbo and Pavlov, Youri I. and Anderson, Karen S. and Tahirov, Tahir H.},
abstractNote = {DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA–DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure–function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA–DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the “fingers” subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Furthermore, pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.},
doi = {10.1074/jbc.RA117.001074},
journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 18,
volume = 293,
place = {United States},
year = {2018},
month = {3}
}

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