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Processing of 3′ Phosphate DNA Termini by the Herpes Simplex Virus‐1 DNA Polymerase: a Novel 3′ Phosphatase Activity
Author(s) -
Boehmer Paul,
Rupesh Kanchi Ravi
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.560.15
Subject(s) - ap endonuclease , ap site , phosphodiester bond , exonuclease , dna polymerase , base excision repair , dna glycosylase , dna , biochemistry , dna ligase , microbiology and biotechnology , chemistry , polymerase , primer (cosmetics) , dna clamp , uracil dna glycosylase , endonuclease , dna repair , biology , polymerase chain reaction , rna , reverse transcriptase , gene , organic chemistry
3′ phosphate terminated DNA ends may arise directly following breakage of the phosphodiester bond or by enzymatic processing of DNA damage including βδ elimination catalyzed by NEIL 1 and NEIL 2 during base excision repair. We have previously shown that HSV‐1 has the capacity to perform uracil‐initiated base excision repair. Specifically, we demonstrated that abasic sites generated by the HSV‐1 UL2 glycosylase are incised by human AP endonuclease and a novel 5′ dRP lyase activity inherent in the UL30 DNA polymerase to generate a 1 nt gap that is subsequently filled by UL30 and ligated by human DNA ligase IIIα/XRCC1 complex. We have explored the ability of UL30 to directly process 3′ phosphate terminated DNA. Our data show that UL30 is able to degrade 3′ phosphate terminated ends of both ssDNA and primer‐templates albeit at lower rates and to a lesser extent than 3′ hydroxyl ends. To determine whether this is due to 3′‐5′ exonuclease or direct 3′ phosphatase, we designed experiments that utilize incorporation of chain terminating dideoxynucleotides to distinguish between exonuclease activity and phosphatase activity. Our data show that UL30 has the ability to extend 3′ phosphate terminated DNA without removal of the terminal nucleotide, indicating an inherent phosphatase activity. Moreover, we show that UL30 is able to directly remove 32 P i from 3′ phosphate terminated DNA, directly demonstrating 3′ phosphatase activity. This allows the enzyme to process 3′ phosphate terminated DNA by direct removal of the phosphate group to permit subsequent nucleotide polymerization. This activity may provide a critical function for the processing of damaged DNA that involves the generation of 3′ phosphate ends to ensure completion of viral DNA replication.