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DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

Parsons JL, Preston BD, O'Connor TR, Dianov GL - Nucleic Acids Res. (2007)

Bottom Line: We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions.Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta).Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK.

ABSTRACT
Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIalpha and DNA polymerase beta. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta). Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol delta are partially deficient in the repair of these 'complex' lesions, demonstrating the importance of Pol delta during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.

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Proposed mechanism of repair of DNA lesions located in close proximity to the 3′-end of a DNA single strand break by Pol δ. Complex DNA strand breaks containing 3′-proximal lesions that are resistant to the major BER enzymes are recognized by Pol δ (step A) that excises the lesion through its associated 3′-5′-exonuclease activity (step B). Pol δ is then able to insert the correct nucleotides into the gap causing strand displacement of the adjacent strand (step C). The subsequent 5′-flap generated is removed by FEN1 and DNA ligase I seals the nick (step D). PCNA can notably stimulate steps C and D.
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Figure 9: Proposed mechanism of repair of DNA lesions located in close proximity to the 3′-end of a DNA single strand break by Pol δ. Complex DNA strand breaks containing 3′-proximal lesions that are resistant to the major BER enzymes are recognized by Pol δ (step A) that excises the lesion through its associated 3′-5′-exonuclease activity (step B). Pol δ is then able to insert the correct nucleotides into the gap causing strand displacement of the adjacent strand (step C). The subsequent 5′-flap generated is removed by FEN1 and DNA ligase I seals the nick (step D). PCNA can notably stimulate steps C and D.

Mentions: Pol δ is one of the major replicative polymerases in chromosomal DNA synthesis although it is also known to be involved in BER (33). The ‘long-patch’ BER pathway involves the incorporation of several nucleotides into the repair gap by Pol δ that cause strand displacement of the adjacent strand creating a flap structure that is excised by the flap endonuclease FEN1 (34,35). This reaction occurs in a PCNA-dependent manner (35) and the fidelity of nucleotide insertion during ‘long-patch’ BER and DNA replication is enhanced by the associated 3′-5′-exonuclease activity of the polymerases (36). However, ‘long-patch’ BER usually occurs as a result of the inability of Pol β to remove the 5′-deoxyribose phosphate moiety following apurinic/apyrimidinic site incision due to modification of the 5′-end by oxidation or reduction. Our study suggests a new important role of Pol δ in the repair of ‘complex’ SSBs. In this scenario the inability of both DNA ligase to seal the DNA strands and of Pol β to ‘move’ the SSB further away from the damaged base by DNA repair synthesis should initiate a switch of repair synthesis to Pol δ. However, damage removal by Pol δ exonuclease activity should precede DNA synthesis (Figure 9).Figure 9.


DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

Parsons JL, Preston BD, O'Connor TR, Dianov GL - Nucleic Acids Res. (2007)

Proposed mechanism of repair of DNA lesions located in close proximity to the 3′-end of a DNA single strand break by Pol δ. Complex DNA strand breaks containing 3′-proximal lesions that are resistant to the major BER enzymes are recognized by Pol δ (step A) that excises the lesion through its associated 3′-5′-exonuclease activity (step B). Pol δ is then able to insert the correct nucleotides into the gap causing strand displacement of the adjacent strand (step C). The subsequent 5′-flap generated is removed by FEN1 and DNA ligase I seals the nick (step D). PCNA can notably stimulate steps C and D.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851633&req=5

Figure 9: Proposed mechanism of repair of DNA lesions located in close proximity to the 3′-end of a DNA single strand break by Pol δ. Complex DNA strand breaks containing 3′-proximal lesions that are resistant to the major BER enzymes are recognized by Pol δ (step A) that excises the lesion through its associated 3′-5′-exonuclease activity (step B). Pol δ is then able to insert the correct nucleotides into the gap causing strand displacement of the adjacent strand (step C). The subsequent 5′-flap generated is removed by FEN1 and DNA ligase I seals the nick (step D). PCNA can notably stimulate steps C and D.
Mentions: Pol δ is one of the major replicative polymerases in chromosomal DNA synthesis although it is also known to be involved in BER (33). The ‘long-patch’ BER pathway involves the incorporation of several nucleotides into the repair gap by Pol δ that cause strand displacement of the adjacent strand creating a flap structure that is excised by the flap endonuclease FEN1 (34,35). This reaction occurs in a PCNA-dependent manner (35) and the fidelity of nucleotide insertion during ‘long-patch’ BER and DNA replication is enhanced by the associated 3′-5′-exonuclease activity of the polymerases (36). However, ‘long-patch’ BER usually occurs as a result of the inability of Pol β to remove the 5′-deoxyribose phosphate moiety following apurinic/apyrimidinic site incision due to modification of the 5′-end by oxidation or reduction. Our study suggests a new important role of Pol δ in the repair of ‘complex’ SSBs. In this scenario the inability of both DNA ligase to seal the DNA strands and of Pol β to ‘move’ the SSB further away from the damaged base by DNA repair synthesis should initiate a switch of repair synthesis to Pol δ. However, damage removal by Pol δ exonuclease activity should precede DNA synthesis (Figure 9).Figure 9.

Bottom Line: We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions.Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta).Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK.

ABSTRACT
Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIalpha and DNA polymerase beta. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta). Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol delta are partially deficient in the repair of these 'complex' lesions, demonstrating the importance of Pol delta during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.

Show MeSH