Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Abstract

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

Figure 1

A schematic illustration of BER subpathways for damaged bases and DNA strand breaks. The damaged base is represented as a star(*). Divergent base excision steps converge to common steps for end processing, followed by repair DNA synthesis (represented as blue dots) and strand sealing. Polβ could be involved in LP-BER by collaborating with FEN1. Other details are discussed in the text.

Figure 2

Distinct end cleaning activities of various enzymes. APE1 removes 3′ blocking PUA (boxed in red in A) while PNK removes 3′ phosphate (boxed in red in B). Polβ removes 5′ blocking dRP (boxed in red in C). Aprataxin hydrolyzes 5′-5′ linkage of AMP to the 5′ phosphate (D). The dRP lyase activity Polβ requires the aldehyde group.

Figure 3

PONDR Plot of the predicted secondary structures of hNEIL1, hNTH1 and their E. coli prototypes, endonuclease VIII (Nei) and endonuclease III (Nth) respectively. The protein sequences were obtained from NCBI database. PONDR. score of 0.5 and higher indicates disordered structure [174]. The disordered C-terminal segment of hNEIL1 and N-terminal segment of hNTH1 are depicted by wiggly lines.