Release of 5′-terminal deoxyribose-phosphate residues from incised abasic sites in DNA by theEscherichia coliRecJ protein

Abstract

Excision of deoxyribose-phosphate residues from enzymatically incised abasic sites in double-stranded DNA is required prior to gap-filling and ligation during DNA base excision-repair, and a candidate deoxyribophosphodiesterase (dRpase) activity has been identified in E. coli. This activity is shown here to be a function of the E. coli RecJ protein, previously described as a 5'-->3' single-strand specific DNA exonuclease involved in a recombination pathway and in mismatch repair. Highly purified preparations of dRpase contained 5'-->3' exonuclease activity for single-stranded DNA, and homogeneous RecJ protein purified from an overproducer strain had both 5'-->3' exonuclease and dRpase activity. Moreover, E. coli recJ strains were deficient in dRpase activity. The hydrolytic dRpase function of the RecJ protein requires Mg2+; in contrast, the activity of E. coli Fpg protein, that promotes the liberation of 5'-->3'Rp residues from DNA by beta-elimination, is suppressed by Mg2+. Several other E. coli nucleases, including exonucleases I, III, V, and VII, endonucleases I, III and IV and the 5'-->3' exonuclease function of DNA polymerase I, are unable to act as a dRpase. Nevertheless, E. coli fpg recJ double mutants retain capacity to repair abasic sites in DNA, indicating the presence of a back-up excision function.

DNA glycosylases in the base excision repair of DNA

“…In the first of these, a 5h-AP endonuclease [8] and a deoxyribophosphodiesterase (dRpase) create a single nucleotide gap, which in eukaryotic cells is filled in by DNA polymerase β and DNA ligase III [9]. This route may also be initiated by bifunctional DNA glycosylases, in which the gap may be created by successive β-and δ-eliminations [10,11]. A role for the presumably noncatalytic XRCC1 (X-ray cross-complementation protein 1) in BER was recently also established.…”

“…Exonuclease III was originally characterized as an exonuclease, but was later shown to be a multifunctional enzyme which probably functions primarily as a 5h AP-endonuclease [17]. The dRpase activity is a property of the recJ gene product [11]. At least one E. coli DNA glycosylase, the formamidopyrimidine-DNA glycosylase (Fpg) protein, may also create a DNA gap alone.…”

“…Further processing requires a 3h-phosphatase, probably contributed by EndoIV [18,19], to produce a primer for DNA polymerase. If an AP site has already been incised by an AP-endonuclease 5h-terminal to the deoxyribose, Fpg also promotes release of deoxyribophosphate [10,11]. A reported dRpase activity associated with E. coli exonuclease I [20] has not been reproduced by others [11].…”

“…If an AP site has already been incised by an AP-endonuclease 5h-terminal to the deoxyribose, Fpg also promotes release of deoxyribophosphate [10,11]. A reported dRpase activity associated with E. coli exonuclease I [20] has not been reproduced by others [11]. Studies with double mutants deficient in RecJ and Fpg have indicated that the dRpase activities of these enzymes are not essential for BER, indicating the presence of a back-up function so far not identified [11].…”

The acidity of uracil and uracil analogs in the gas phase: Four surprisingly acidic sites and biological implications

“…Hydrogen bonding modulates recognition of DNA and RNA bases, and the interaction energy between two bonded complementary nucleobases is dependent on the intrinsic basicity of the acceptor atoms as well as on the acidity of donor NH groups [1,2]. In addition, understanding the intrinsic reactivity of nucleic bases can shed light on key biosynthetic mechanisms in which nucleobases are substrates [3][4][5][6][7][8].…”