TY - JOUR
T1 - Structural Insights into Phosphorylation-Mediated Polymerase Function Loss for DNA Polymerase β Bound to Gapped DNA
AU - Srivastava, Amit
AU - Idriss, Haitham
AU - Homouz, Dirar
N1 - Publisher Copyright:
© 2023 by the authors.
PY - 2023/5
Y1 - 2023/5
N2 - DNA polymerase (Formula presented.) is a member of the X-family of DNA polymerases, playing a critical role in the base excision repair (BER) pathway in mammalian cells by implementing the nucleotide gap-filling step. In vitro phosphorylation of DNA polymerase (Formula presented.) with PKC on S44 causes loss in the enzyme’s DNA polymerase activity but not single-strand DNA binding. Although these studies have shown that single-stranded DNA binding is not affected by phosphorylation, the structural basis behind the mechanism underlying phosphorylation-induced activity loss remains poorly understood. Previous modeling studies suggested phosphorylation of S44 was sufficient to induce structural changes that impact the enzyme’s polymerase function. However, the S44 phosphorylated-enzyme/DNA complex has not been modeled so far. To address this knowledge gap, we conducted atomistic molecular dynamics simulations of pol (Formula presented.) complexed with gapped DNA. Our simulations, which used explicit solvent and lasted for microseconds, revealed that phosphorylation at the S44 site, in the presence of Mg ions, induced significant conformational changes in the enzyme. Specifically, these changes led to the transformation of the enzyme from a closed to an open structure. Additionally, our simulations identified phosphorylation-induced allosteric coupling between the inter-domain region, suggesting the existence of a putative allosteric site. Taken together, our results provide a mechanistic understanding of the conformational transition observed due to phosphorylation in DNA polymerase (Formula presented.) interactions with gapped DNA. Our simulations shed light on the mechanisms of phosphorylation-induced activity loss in DNA polymerase (Formula presented.) and reveal potential targets for the development of novel therapeutics aimed at mitigating the effects of this post-translational modification.
AB - DNA polymerase (Formula presented.) is a member of the X-family of DNA polymerases, playing a critical role in the base excision repair (BER) pathway in mammalian cells by implementing the nucleotide gap-filling step. In vitro phosphorylation of DNA polymerase (Formula presented.) with PKC on S44 causes loss in the enzyme’s DNA polymerase activity but not single-strand DNA binding. Although these studies have shown that single-stranded DNA binding is not affected by phosphorylation, the structural basis behind the mechanism underlying phosphorylation-induced activity loss remains poorly understood. Previous modeling studies suggested phosphorylation of S44 was sufficient to induce structural changes that impact the enzyme’s polymerase function. However, the S44 phosphorylated-enzyme/DNA complex has not been modeled so far. To address this knowledge gap, we conducted atomistic molecular dynamics simulations of pol (Formula presented.) complexed with gapped DNA. Our simulations, which used explicit solvent and lasted for microseconds, revealed that phosphorylation at the S44 site, in the presence of Mg ions, induced significant conformational changes in the enzyme. Specifically, these changes led to the transformation of the enzyme from a closed to an open structure. Additionally, our simulations identified phosphorylation-induced allosteric coupling between the inter-domain region, suggesting the existence of a putative allosteric site. Taken together, our results provide a mechanistic understanding of the conformational transition observed due to phosphorylation in DNA polymerase (Formula presented.) interactions with gapped DNA. Our simulations shed light on the mechanisms of phosphorylation-induced activity loss in DNA polymerase (Formula presented.) and reveal potential targets for the development of novel therapeutics aimed at mitigating the effects of this post-translational modification.
KW - DNA polymerase β
KW - MD simulations
KW - post-translational modifications
KW - principal component analysis
UR - http://www.scopus.com/inward/record.url?scp=85160376912&partnerID=8YFLogxK
U2 - 10.3390/ijms24108988
DO - 10.3390/ijms24108988
M3 - Article
C2 - 37240334
AN - SCOPUS:85160376912
SN - 1661-6596
VL - 24
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 10
M1 - 8988
ER -