Base excision repair capacity in informing healthspan

Abstract

Base excision repair (BER) is a frontline defense mechanism for dealing with many common forms of endogenous DNA damage, several of which can drive mutagenic or cell death outcomes. The pathway engages proteins such as glycosylases, abasic endonucleases, polymerases and ligases to remove substrate modifications from DNA and restore the genome back to its original state. Inherited mutations in genes related to BER can give rise to disorders involving cancer, immunodeficiency and neurodegeneration. Studies employing genetically defined heterozygous (haploinsufficient) mouse models indicate that partial reduction in BER capacity can increase vulnerability to both spontaneous and exposure-dependent pathologies. In humans, measurement of BER variation has been imperfect to this point, yet tools to assess BER in epidemiological surveys are steadily evolving. We provide herein an overview of the BER pathway and discuss the current efforts toward defining the relationship of BER defects with disease susceptibility.

Fig. 1.

Short-patch BER pathway. A DNA glycosylase excises an aberrant base (e.g. uracil = U or thymine glycol = Tg). Monofunctional glycosylases create an AP site, which is incised by APE1 (left), whereas bifunctional glycosylases cleave the DNA backbone at the AP site via β-elimination or β,δ-elimination, creating a 3′-terminus that must be removed by APE1 (center) or polynucleotide kinase/phosphatase (PNKP) (right), respectively. Following end-cleanup (highlighted in red), single-nucleotide repair synthesis is conducted by POLβ and nick ligation is carried out by an XRCC1/LIG3α complex (highlighted in blue). See text for additional details.

Fig. 2.

Long-patch BER pathway. The black star represents a 5′ end modification that is not a substrate for the lyase activity of POLβ. In this scenario, or in situations of low intracellular ATP concentration where ligation is impeded, strand-displacement synthesis is carried out by POLβ, or one of the replication-associated polymerases, POLε or POLδ, to generate a 5′ flap structure. This structure is processed by FEN1, and repair is completed, likely in cooperation with its interacting protein partners proliferating cell nuclear antigen (PCNA) and LIG1.

Fig. 3.

Single-step BER assays. (A) Radiolabeled substrate assay. The synthetic site-specific, damage-containing (primarily a base modification or AP site) oligonucleotide is end-labeled (green star) and annealed to a complementary unlabeled strand. The duplex substrate is incubated with a protein extract, and the strand cleavage (repair) efficiency is quantified following separation of substrate (S) and product (P) on a denaturing polyacrylamide gel (right). (B) Molecular beacon assay. A damage-containing substrate (depicted as a stable hairpin structure) is synthesized to harbor both a fluorophore (yellow star) and a compatible quench (black dot). Following processing by repair enzymes (intermediate product in brackets), the short product spontaneously dissociates and the fluorophore signal is measured by a plate reader (in vitro) or microscopy (in vivo). (C) The solid support microarray assay. This method, which works off a similar excision/incision principle to the assays above, measures the ability of an extract to reduce the fluorescent signal by processing an affixed damage-containing DNA substrate. Following treatment (step 1) and subsequent washing steps (step 2), the percentage of fluorophore remaining bound to the solid support is quantified to determine efficiency of repair. See Table II for further details about these assays.

Fig. 4.

BER pathway assays: (A) Radiolabeled nucleotide incorporation assay. An unlabeled damage-containing DNA substrate (S; oligonucleotide substrate depicted) is incubated with a protein extract in the presence of a radiolabeled nucleotide (red bar-green star). BER processing results in the formation of radiolabeled repair intermediates (P1) and the radiolabeled fully repaired product (P2), which can be visualized following resolution on a denaturing polyacrylamide gel (right). (B) HCR assay. As depicted, the damage introduced blocks transcription of the reporter gene, resulting in low signal following transfection. Upon repair, the reporter gene signal is restored, allowing quantification of luciferase activity or fluorescence by plate reader technologies. (C) Comet assay. Cells are harvested with or without DNA-damaging agent treatment, and the level of chromosome damage is quantified by embedding single cells into an agarose gel, lysing them and subjecting their DNA to electrophoresis. A greater comet tail relative to the comet head is reflective of a higher level of DNA damage. See Table II for further details about these assays. HCR, host cell reactivation.