4. ENDOGENOUS DNA DAMAGE, MUTAGENESIS AND AGING
One of the more attractive theories offered to explain aging involves the gradual accumulation of mutations in genomic DNA. Mutations arise from DNA damage generated in part by products of cellular metabolism when the rate of DNA damaging events exceeds the rate of lesion repair. Among the most common forms of endogenous DNA damage are modified bases generated by reactive oxygen species (1-3), abasic sites produced by spontaneous or enzymatic release of bases from DNA (4, 5) and exocyclic DNA adducts formed from products of lipid peroxidation (6). In this chapter, we describe the miscoding and mutagenic potential of some of these lesions.
Cellular oxidation is a major contributor to endogenous DNA damage. The ubiquitous presence of reactive oxygen species ensures that 8-oxydeoxyguanosine (8-oxodG) exists in the DNA of all living organisms. The mutagenic potential of 8-oxodG is reflected in its miscoding properties; namely, dAMP is incorporated opposite the modified base during translesional synthesis (8). Striking differences among DNA polymerases were observed with respect to the relative amounts of dAMP and dCMP incorporated opposite the lesion in vitro (Table 4.I). These differences play an important role in the cellular repair of 8-oxodG in DNA (9). Plasmid vectors, modified site-specifically, have been used to establish the mutagenic potential of 8-oxodG in cells (10-13). Oligodeoxynucleotides containing a single modified base were ligated to a single stranded (ss) DNA shuttle vector and allowed to replicate in E. coli and simian kidney (COS7) cells.
The number of transformants obtained determined the blocking effect of the lesion.
Relative incorporation of nucleotides opposite 8-oxoG by DNA polymerase in vitro1
1Data from Shibutani et al. (8) and unpublished experiments
|DNA polymerase||Incorporation C:A|
Progeny DNA was recovered from host cells and the base sequence in the vicinity of the lesion determined. The predominant mutations induced by 8-oxodG in bacteria and mammalian cells were G—>T transversions, targeted to the site of the lesion. This finding is consistent with results of primer-extension studies conducted in vitro (8). The mutational frequency for 8-oxodG in E. coli and in COS cells is less than 3%; thus, the modified base is weakly mutagenic in these experimental systems (13). The miscoding and mutagenic properties of 8-oxodG are reflected in the structure of duplex DNA containing this lesion. When 8-oxodG is positioned opposite dA, the modified base assumes the syn conformation, forming a Hoogstein base pair (15-16) (Fig. 4.1). Furthermore, the dimensions of the 8-oxodG:dA pair approximate Watson-Crick geometry, shielding the incoming base from proofreading and permitting efficient extension of the nascent chain from the 3' primer terminus (8).
In this respect, 8-oxodG differs from the structurally related lesion, formamidopyrimidine (Fapy), which effectively blocks chain extension (17) and diminishes its mutagenic potential.
Fig.4.1. Base pairing for 8-oxodG (syn): dA and 8-oxodG (anti): dC
Fig. 4.2. Structures of natural and synthetic abasic sites
Mutator strains of E. coli illustrate the role of DNA repair enzymes in protecting this organism against the mutagenic effects of 8-oxoguanine (9, 18). MutT strains of E. coli unable to hydrolyze 8-oxoGTP (19) are characterized by a high frequency of A:T—>C:G transversions. MutM strains deficient in Fpg protein (8-oxoguanine DNA glycosylase) (20) and MutY strains deficient in adenine DNA glycosylase (21) exhibit elevated levels of G:C—>T:A transversions. MutM/MutY double mutants act synergistically, indicating that products of mutM and mutY combine to protect E. coli from the mutagenic effects of 8-oxodG (22). Functional homologs of mutM, mutY, and mutt have been identified in mammalian cells (23-26); it is likely that a similar error avoidance pathway operates in humans.