Abstract
Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. Deinococcus radiodurans, an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from D. radiodurans and found that it possesses AP endonuclease, 3′-phosphodiesterase, 3′-phosphatase, and 3′–5′ exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg2+ and Mn2+ were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the drxth gene in D. radiodurans caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.
Highlights
Genomic DNA regularly suffers accidental damage caused by endogenous and exogenous agents
Sequence alignment suggested that dr_0354 encodes a unique AP endonuclease that is homologous to E. coli exonuclease III (Supplementary Figure S2)
To confirm the biochemical activity of the DrXth protein, we first tested AP endonuclease activity using the 5′-FAM-labeled oligonucleotide duplex THF.T containing an abasic site in the middle of the DNA duplex
Summary
Genomic DNA regularly suffers accidental damage caused by endogenous and exogenous agents. One of the most abundant types of damage is apurinic/apyrimidinic (AP) sites, resulting from spontaneous water-mediated depurination or depyrimidination, or hydrolysis of the N-glycosyl bond between the base and the sugar-phosphate backbone by DNA glycosylase (Wilson and Barsky, 2001). AP sites are cytotoxic and increase mutagenesis through deletions, insertions, or substitutions if bypassed by DNA polymerases (Loeb and Preston, 1986). The major contribution to AP sites cleavage is provided by AP endonuclease, a crucial enzyme in the base excision repair (BER) pathway (Wilson and Barsky, 2001). AP endonuclease incises DNA on the 5′ side of the abasic site, leaving a 3′-terminal
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