Noncognate DNA damage prevents the formation of the active conformation of the Y‐family DNA polymerases DinB and DNA polymerase κ

Abstract

Y-family DNA polymerases are specialized to copy damaged DNA and are associated with increased mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have to date not been extensively characterized. Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2-furfuryl-deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6-methyl-deoxyguanosine that is a poor substrate. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved pre-chemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N terminus, alters helix dynamics, and reduces DNA affinity

“…This work is one of the few examples utilizing HDX to study protein-DNA interactions (33,34), and the only one that we are aware of to examine the effects of metal and DNA binding on protein dynamics. According to previous studies employing X-ray absorption spectroscopy as a structural probe and LacZ assays to assess function (18,29,32), Co(II) and Ni(II) bind to RcnR with slightly different ligand sets, employing His-3 for Co(II) but not for Ni(II) (29).…”

Mammalian DNA Polymerase Kappa Activity and Specificity

“…The palm domain in DNA polymerases is associated with catalysis [2]; however, regions in the palm, thumb, and little finger domain were observed to participate in this conformational change. These regions are distally located to the active site, and some do not make contact with the incoming dNTP [43]. This work not only demonstrates the importance of more distal regions of the protein and their ability to discriminate between the correct and incorrect incoming nucleotide but also provides evidence for a fidelity checkpoint in pol κ.…”

“…The crystal structures of pol κ generally show a large conformational change upon binding DNA, but comparison of crystal structures of binary versus ternary structures are relatively similar regardless of dNTP identity [21,22]. However, kinetics, thermal stability assays, and hydrogen–deuterium exchange-mass spectrometry (HDXMS) experiments indicate that pol κ adopts a specific, active conformation only when the correct or preferred substrates are bound, i.e., undamaged DNA or N 2 -furfuryl-dG (minor groove damage)-containing templates and the correct incoming dNTP [43,44]. The palm domain in DNA polymerases is associated with catalysis [2]; however, regions in the palm, thumb, and little finger domain were observed to participate in this conformational change.…”

Association Between Single Nucleotide Polymorphisms in DNA Polymerase Kappa Gene and Breast Cancer Risk in Chinese Han Population

“…On the other hand, low fidelity of DNA replication is necessary for the evolution of species, for generating diversity that can lead to increased survival and adaptability of species in the changing environment. 10 , 11 TLS, which largely depends on specialized DNA polymerases, repairs and replicates damage DNA with low fidelity. 12 Y-family DNA polymerases are specialized polymerases involved in TLS, replicating damaged DNA, bypassing damaged nucleotides that block the normal progression of replication forks.…”