Molecular snapshots of APE1 proofreading mismatches and removing DNA damage

Abstract

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair enzyme which uses a single active site to process DNA damage via two distinct activities: (1) AP-endonuclease and (2) 3′ to 5′ exonuclease. The AP-endonuclease activity cleaves at AP-sites, while the exonuclease activity excises bulkier 3′ mismatches and DNA damage to generate clean DNA ends suitable for downstream repair. Molecular details of the exonuclease reaction and how one active site can accommodate various toxic DNA repair intermediates remains elusive despite being biologically important. Here, we report multiple high-resolution APE1–DNA structural snapshots revealing how APE1 removes 3′ mismatches and DNA damage by placing the 3′ group within the intra-helical DNA cavity via a non-base flipping mechanism. This process is facilitated by a DNA nick, instability of a mismatched/damaged base, and bending of the DNA. These results illustrate how APE1 cleanses DNA dirty-ends to generate suitable substrates for downstream repair enzymes.

APE1: A skilled nucleic acid surgeon

“…This substrate overlap implies a level of evolved redundancy among enzymes that may play a particular biological role during different cellular stages and in response to varying types of DNA damage. Despite this ambiguity and overlap, the biological relevance of the APE1 exonuclease activity has been identified in several instances, including: (1) APE1 removal of 3ʹ α,β-unsaturated aldehyde (PUA) groups resulting from bi-functional glycosylases during BER [ 54 ]; (2) the cleansing of oxidatively damaged DNA dirty ends, such as 3ʹ PG (phosphoglycolate) and 3ʹ 8-oxoG (8-oxoGuanine) by APE1 both in vitro and in cellular extracts [ 55 , 56 ]; (3) APE1 proofreading of DNA ligation confounding misinsertions by DNA polymerase β during BER, [ 11 , [57] , [58] , [59] ]; and (4) the association between APE1 variants with reduced exonuclease activity and carcinogenesis [ [60] , [61] , [62] ]. Below, we will discuss intriguing biological models for some of these APE1 scenarios and how one protein active site can accommodate an array of nucleic acid substrates.…”

“…Substrates for end processing enzymes are indicated in parentheses. Enzyme k cat /K M (s −1 μM -1 ) Reference End Processing Enzymes APE1 (3'-PUA) 2.7 [ 54 ] APE1 (3'-Mismatch) 1 [ 1 , 11 ] APE2 (3ʹ-Mismatch) 4 × 10 −6 [ 107 ] PNK (3'-Phosphate) 30 [ 54 ] Tdp1 (3′-Phosphotyrosine) 0.25 [ 108 ] Aprataxin (5'-AMP) 3 [ 109 ] Pol β (5ʹ-dRP lyase) 0.15 [ 110 ] BER Enzymes UNG 500 [ 110 ] OGG1 0.03 [ 110 ] APE1 (AP-DNA) 100 [ 110 ] pol β (insertion) 1.5 [ 110 ] Pol β (5ʹ-dRP lyase) 0.15 [ 110 ] Lig I 0.4 [ ...…”

“…2 B) is perplexing based on the APE1 AP-endonuclease structures alone. As a result, its precise biological relevance has been difficult to ascertain and the literature contains conflicting opinions on the mechanistic details of the APE1 exonuclease activity [ 11 , 35 , 39 ].…”

“…To better understand how APE1 accommodates mismatched nucleotides during the exonuclease activity, our group recently published X-ray crystal structures of APE1 in complex with a C/T mismatch [ 11 ]. This substrate represents a putative BER intermediate in which polymerase β has incorrectly inserted a C across from a templating T. Then, APE1 could enact its exonuclease activity to remove the 3′ misinserted C. We chose a C/T mismatch because data collected in our lab, as well as previously published by others, indicates that mismatches containing C represent optimal substrates for the APE1 exonuclease activity [ [69] , [70] , [71] ].…”

“…Due to a wealth of structural and biochemical data, it is well understood how APE1 endonucleolytically cleaves at AP sites during base excision repair (BER) [ 1 , 9 , 10 ]. Recent X-ray crystal structures have revealed the fundamentals of the 3ʹ to 5ʹ exonuclease mechanism, which can function to proofread 3ʹ mismatches inserted by DNA polymerase β during BER and remove 3ʹ DNA end damage at single strand breaks [ 11 ]. This newly uncovered mechanism used by APE1 for its exonuclease activity provides new insights into the strategies used by APE1 to cleave during some of its less-studied nuclease activities, such as those utilized during nucleotide incision repair (NIR) and RNA metabolism.…”