Format

Send to

Choose Destination
DNA Repair (Amst). 2016 Oct;46:20-28. doi: 10.1016/j.dnarep.2016.08.002. Epub 2016 Sep 1.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

Author information

1
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: tokarsky.8@osu.edu.
2
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: gadkari.3@osu.edu.
3
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: zahurancik.2@osu.edu.
4
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA. Electronic address: malikchanchal@gmail.com.
5
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA. Electronic address: ashis.basu@uconn.edu.
6
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: suo.3@osu.edu.

Abstract

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.

KEYWORDS:

3-Nitrobenzanthrone; DNA repair; DNA replication; Human DNA polymerase eta; Translesion synthesis

PMID:
27612622
PMCID:
PMC5048595
DOI:
10.1016/j.dnarep.2016.08.002
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center