Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

Olga Rechkoblit,Satya Prakash,Yogesh K Gupta,Louise Prakash,Aneel K Aggarwal,Robert E Johnson

doi:10.1038/s41467-021-24317-z

Abstract

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

Highlights

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine via translesion synthesis, in mostly error-free manner
PrimPol is the only human DNA polymerase that is known to localize to mitochondria and is able to efficiently and mostly accurately bypass an oxoG via translesion synthesis (TLS)[21,22,23,24,25]
To produce the insertion ternary PrimPol complexes with the correct C incoming nucleotide triphosphate opposite the oxoG lesion, we crystallized the catalytic core of human PrimPol with a 17-nucleotide DNA template (5′-CA(oxoG)CGCTACCACACCCC-3′) and a 2′deoxy-3′-terminated 12 nt DNA primer (5′-GGTGTGGTAGCG3′) in the presence of dCTP

Summary

Results

To produce the insertion ternary PrimPol complexes with the correct C incoming nucleotide triphosphate opposite the oxoG lesion, we crystallized the catalytic core of human PrimPol (residues 1−354) with a 17-nucleotide (nt) DNA template (5′-CA(oxoG)CGCTACCACACCCC-3′) and a 2′deoxy-3′-terminated 12 nt DNA primer (5′-GGTGTGGTAGCG3′) in the presence of dCTP. To capture the extension step we used a 17 nt DNA template (5′-CAT(oxoG)CCTACCACACCCC–3′), where the oxoG lesion is moved one base downstream compared to its location in the insertion complex, and 13 nt DNA primers with either a C or an A 2′-deoxy-3′-terminal bases (5′GGGTGTGGTAGGX-3′, where X is C or A), and the correct incoming base dATP. The template oxoG residue (oxoGdCTP) is refined to 2.60 Å resolution and is similar to the structure of the unmodified complex (T-dATP) (root mean square deviation (rmsd) of ~0.35 Å over 249 Cαs of molecule A complexes)

G12 Primer

Discussion

G74 T3 N289

A14 A14 in molecule A

Methods