TY - JOUR
T1 - Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells
AU - Yoon, Jung Hoon
AU - Johnson, Robert E.
AU - Prakash, Louise
AU - Prakash, Satya
N1 - Funding Information:
This study was supported by National Institutes of Health Grants ES022948 and GM126087. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Yoon et al.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethe-nodeoxyadenosine (εdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through εdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize εdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.
AB - The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethe-nodeoxyadenosine (εdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through εdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize εdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.
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U2 - 10.1074/jbc.RA120.012816
DO - 10.1074/jbc.RA120.012816
M3 - Article
C2 - 32169903
AN - SCOPUS:85084395008
SN - 0021-9258
VL - 295
SP - 5918
EP - 5927
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -