Visualizing Rev1 catalyze protein-template DNA synthesis

Abstract

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

“…A recent time-lapse crystallographic study of yeast Rev1 found that the ejection of the template base preceded nucleotide binding and that the templating base remained extrahelical even after nucleotide incorporation ( 12 ). Hydrogen bonding between the backbone amides of M685/G686 in the G-loop of yeast Rev1 help keep the template base extrahelical.…”

Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC

“…Slow PP i release in the absence of the next correct dNTP is an intrinsic property of PolC, as it does not depend on association with the β-clamp, nor does it depend on either the N-terminal or exonuclease domains of the polymerase ( 25 ). Steady-state analysis of two Y-family DNA polymerases suggest that PP i is hydrolyzed at the polymerase active site prior to release ( 43 , 44 ). However, this is clearly not a general phenomenon as PolC does not hydrolyze PP i to Pi on a timescale that is relevant to nucleotide incorporation ( Supplementary Figure S5A ).…”

Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus

“…S. cerevisiae and human Rev1 function as a dCMP transferase, incorporating strictly dCMP opposite abasic sites and dG-lesions ( Nelson et al, 1996 ; Lin et al, 1999 ; Masuda et al, 2001 ). dCMP transferase activity is instructed by Rev1’s conserved active site side chain arginine residue via a protein-templated mechanism ( Nair et al, 2005 ; Weaver et al, 2020 ). We hypothesized that the conservation of the functional arginine and Rev1 consensus sequence ( Figure 7A ) would make T. lanuginosis Rev1 a bonafide dCMP transferase.…”

“…The unique feature of eukaryotic Rev1 orthologs is their efficiency at bypassing both damaged guanines and abasic sites using a deoxycytidyl transferase mechanism that limits Rev1 to exclusively incorporate dC ( Nelson et al, 1996 ). This is achieved by displacing the DNA lesion from the active site entirely and instead using a protein sidechain (R324 in S. cerevisiae and R357 in H. sapiens ) as the “template” which base pairs solely with dC ( Nair et al, 2005 , 2011 ; Weaver et al, 2020 ).…”