The two major systems for the repair of ultraviolet (UV) radiation-induced DNA damage in cells are nucleotide excision repair (Petit and Sancar, 1999), and recombinational DNA repair (Kuzminov, 1999; Smith, 2004). In nucleotide excision repair, damaged nucleotides (i.e., pyrimidine dimers) are recognized and cut out, and the resulting hole is patched by DNA polymerase l, using the DNA strand opposite the gap as the template. The patch is then joined to the main strand by DNA ligase. This process is very accurate, and does not produce mutations. The sheer volume of publications on "cut and patch" nucleotide excision repair (e.g., Petit and Sancar, 1999) has generated the impression that cells possess only this simple repair system.
Quite to the contrary, recombinational DNA repair is critical for the survival of UV radiation-damaged cells. It accounts for about 50% of the survival of UV irradiated Escherichia coli (see below). It is a very complicated process that requires two DNA duplexes, and the exchange of a strand of DNA from one DNA duplex to the other (Rupp et al., 1971; Howard-Flanders and Rupp, 1972; Ganesan, 1974; Wang and Smith, 1984), and it produces mutations.
The first indication that nucleotide excision repair ("cut and patch") is NOT the only mechanism by which cells repair damage to their DNA, was the observation that bacterial cells deficient in nucleotide excision repair (i.e., uvrA) or in genetic recombination (i.e., recA) are very sensitive to UV radiation, and show a similar level of survival after UV irradiation. A double mutant (uvrA recA), however, is much more sensitive to UV irradiation than either of the single mutants (Figure 1). From the most fundamental principles of radiation biology and genetics, these data argue that, (a) these two systems, i.e., coded by the uvrA and the recA genes, function largely independently of each other, and (b) they are of about equal importance to the survival of UV-irradiated cells of E. coli K-12. These studies led to the discovery of postreplication repair (see below).
Figure 1. UV radiation survival curves for DNA repair deficient mutants of E. coli K-12. The uvrA6 mutation blocks nucleotide excision repair, and the recA13 mutation blocks recombinational DNA repair. Note that the double mutant, uvrA6 recA13 is very much more sensitive to UV radiation than either of the single mutants, indicating that the two single mutants are involved in separate pathways of DNA repair. Since the two single mutants have about the same sensitivity, it indicates that nucleotide excision repair and recombinational DNA repair are of about equal importance to the survival of UV irradiated E. coli. [Modified from Howard-Flanders and Boyce, 1966]
Figure 2. Schematic of DNA replication in E. coli with lesions (large dots), both in the DNA that was replicated prior to UV irradiation, where two DNA duplexes exist, and in that portion of the chromosome prior to replication, where only one DNA duplex exists. The problems and opportunity for recombinational DNA repair in these two regions of the chromosome are markedly different.
The DNA synthesized immediately after UV irradiation in excision repair-deficient cells (and also wild-type cells; see below) of E. coli K-12 has discontinuities when assayed in alkaline sucrose gradients. The mean length of newly synthesized DNA approximates the distance between pyrimidine dimers in the parental strand. With further incubation of the cells, however, these discontinuities disappear, and the DNA approximates the molecular size of that from unirradiated control cells (Rupp and Howard-Flanders, 1966; Howard-Flanders et al., 1968). The exchanges envisioned by this type of repair resemble those involved in genetic recombination (Rupp et al., 1971; Rupp and Howard-Flanders, 1968). This prediction has been verified by demonstrating that recA cells are deficient in the production of normal length DNA from the small pieces of DNA synthesized immediately after UV irradiation (Smith and Meun, 1970; Sedgwick, 1975).
When DNA synthesis proceeds along a damaged template, synthesis halts at the site of a non-coding lesion, and then resumes downstream from the lesion (i.e., at the next DnaG primase-binding site), leaving gaps in the newly synthesized daughter strand opposite the UV radiation-induced lesion in the parental strand (Rupp and Howard-Flanders, 1968). The fact that photoreactivation after UV irradiation in a uvrA strain stimulated gap filling, is taken as further evidence that a large proportion of the DNA daughter-strand gaps are opposite pyrimidine dimers (Bridges and Sedgwick, 1974). [see Photoreactivation module]
The dimers that are opposite DNA daughter-strand gaps are no longer subject to excision, since this process requires an intact complementary strand (Jansz, Pouwels and Van Rotterdam, 1963; Yarus and Sinsheimer, 1984). Only after the gaps are filled by sister-strand exchanges will the dimers again be subject to excision repair.
These gaps in the daughter strands, which average 1000 nucleotides in length (Iyer and Rupp, 1971), are subsequently repaired in recombination proficient strains by transferring the appropriate sections of DNA from the parental strands into the daughter strands. This transfer of parental strands into daughter strands has been confirmed by direct measurement (Rupp et al., 1971; Howard-Flanders and Rupp, 1972; Ganesan, 1974; Wang and Smith, 1984). Although most studies on postreplication repair have been performed in excision repair deficient cells, this type of repair is fully operative in wild-type cells (Smith and Meun, 1970; Rupp, Iyer and Zipser, 1973; Howard-Flanders and Rupp, 1981).
Although postreplication repair (i.e., the repair of DNA daughter-strand gaps) is completely dependent upon the recA gene, mutations in the recB and recC genes do NOT cause a deficiency in the repair of DNA daughter-strand gaps (Smith and Meun, 1970). However, the recB gene is known to function in the repair of DNA double-strand breaks that are formed metabolically after UV irradiation in E. coli (Wang and Smith, 1975). In fact, unrepaired DNA double-strand breaks appear to be the major cause of lethality in UV-irradiated wild-type bacteria (Bonura and Smith, 1975a, b). The repair of metabolically-produced DNA double-strand breaks constitutes a second type of recombination repair that is distinct from the repair of DNA daughter-strand gaps, i.e., it is recBC-dependent (Wang and Smith, 1975, 1986). [see module on
DNA Double-Strand Breaks]
Multiple Pathways of Postreplication Repair
Three pathways are known for the repair of DNA daughter-strand gaps, i.e., the recF-dependent, the recF-independent, and the umuCD-dependent pathways. Much of postreplication repair is constitutive (Ganesan and Smith, 1972; Sedgwick, 1975), but a portion (i.e., umuCD) is inducible by UV radiation, and is responsible for UV radiation mutagenesis (see below). Each of these pathways is recBC-independent (Smith and Meun, 1970).
RecF Pathway: About half of the DNA daughter-strand gaps are repaired by a recF-dependent process (Wang and Smith, 1975; Ganesan and Seawell, 1975; Kato, 1977; Tseng, Hung and Wang, 1994). The involvement of the recF gene suggests that the recF pathway of homologous recombination may be involved in this repair process. The RecF protein is one of at least three single-strand DNA binding proteins, along with the RecA and Ssb proteins (Madiraju and Clark, 1991).
The repair of daughter-strand gaps by the recF-dependent and the recF-independent process (see below) is accompanied by the transfer of DNA lesions from the parental strand to the daughter strand (Ganesan, 1974; Wang and Smith, 1984). This occurs about 50% of the time in E. coli (Ganesan, 1974), and appears to be due to the random resolution of the Holliday junction (e.g., Sigal and Alberts, 1972), an intermediate in recombination.
RecF-Independent Pathway: The fact that a uvrB recF stain is not as deficient in the repair of daughter-strand gaps as is a uvrB recA strain suggested that a second pathway must exist for the repair of daughter-strand gaps (Wang and Smith, 1975). This conclusion was supported by studies using an insertion mutation of recF (recF332::Tn3) to ensure that the earlier results were not due to leakiness in the original recF143 mutation. The recF-independent pathway is also independent of the recBC genes, and is constitutive (Sharma and Smith, 1985). Studies using polA mutants, indicate that the polA gene (DNA polymerase l) plays a major role in the recF-independent repair of daughter-strand gaps. Studies on different polA mutants (i.e., polA1, polAex2, polA, etc.) suggest that it is the 5'3' exonuclease activity of DNA polymerase l that plays a major role in the repair of daughter-strand gaps (Sharma and Smith, 1987).
Furthermore, since DNA polymerase is known to be involved in the joining of Okazaki fragments synthesized in the lagging strand of unirradiated cells, this raises the possibility that the daughter-strand gaps formed in the lagging strand of UV irradiated cells may be selectively repaired by the recF-independent, polA-dependent pathway, while the daughter-strand gaps formed in the leading strand (i.e., presumably longer gaps) may be repaired by the recF-dependent pathway (Liu, Cheng and Wang, 1998).
UmuC Pathway: Since a uvrA polA recF strain is not quite as deficient in the repair of daughter-strand gaps as is a uvrA recA strain (Sharma and Smith, 1987), it suggests that a third pathway must exist for the repair of daughter-strand gaps. Consistent with this observation, a small fraction of the repair of daughter-strand gaps is dependent upon the umuC gene, but is independent of the recF and recBC genes (Wang and Smith, 1985). A uvrA polA recF umuC strain has not yet been tested to see if it as deficient as a uvrA recA strain in the repair of daughter-strand gaps.
The UmuC and UmuD proteins combine, after the selective cleavage of the UmuD protein by RecA, to form an error-prone polymerase (UmuD'2UmuC), polV (Tang et al., 1999; Ferentz, Walker and Wagner, 2001), which can synthesize past lesions in DNA. This is consistent with the fact that umuC controls all of UV radiation mutagenesis (Kato and Shinoura, 1977). A umuC mutation, however, has only a partial effect on spontaneous mutagenesis (Sargentini and Smith, 1981), and on X-ray mutagenesis (Sargentini and Smith, 1989). [see module on
UV Radiation and Spontaneous Mutagenesis]
Nucleotide Excision Repair
There are two pathways of nucleotide excision repair. One pathway is DNA polymerase l dependent, growth medium independent (i.e., macromolecular synthesis is not required), and it produces short repair patches (about 20 nucleotides long). This pathway requires only one DNA duplex (Petit and Sancar, 1999).
The second excision repair process, long-patch excision repair, which requires two DNA duplexes, is largely ignored by reviewers (e.g., Hanawalt, 2001). Nevertheless, this excision repair pathway does exist, and it has been confirmed by other authors (e.g., Youngs et al., 1974). It is dependent upon the recA gene, it is growth medium dependent (i.e., macromolecular synthesis is required), and it produces long repair patches (1500-9000 nucleotides long) (Cooper and Hanawalt, 1972a, b; Cooper, 1982). Long-patch excision repair also requires the recF gene (Hanawalt et al., 1982), but does NOT require the recBC genes (Hanawalt, Cooper and Smith, 1981).
When wild-type cells are allowed to repair their DNA after UV irradiation in the presence of chloramphenicol to inhibit the synthesis of induced proteins, only about 80% of the dimers are excised (Lin, Kovalsky and Grossman, 1997). Similarly, a recA mutant, which is deficient in the induction of proteins after UV irradiation, only excises about 80% of the dimers compared to a wild-type strain (Shlaes, Anderson, and Barbour, 1972). The early repair seems to be short-patch excision repair, which occurs immediately after UV irradiation, and is controlled by DNA polymerase l (Cooper and Hanawalt, 1972a), while the induced repair appears to be the long-patch system that is controlled by recA (Cooper, 1982). Additional copies of the UvrA protein (Kenyon and Walker, 1981) and the UvrB protein (Schendel, Fogliano, and Strausbaugh, 1982) are synthesized after UV irradiation, and may be relevant to the inducible long-patch excision repair process.
The excision repair that occurs in cells that contain completely replicated chromosomes, i.e., where only one DNA duplex is present per chromosome, is not dependent upon recA. In this situation, classical nucleotide excision repair occurs, i.e., without strand exchanges. The excision repair that functions in the part of the chromosome that was replicated before UV irradiation (i.e., where two DNA duplexes exist, Figure 1), is recA-dependent (Smith and Sharma, 1987).
The similarities between the genetic requirements for long-patch excision repair and the repair of DNA daughter-strand gaps, i.e., the requirement for recA and recF, but not recBC, and the requirement for sister DNA duplexes, suggests that the mechanisms for these two repair processes are similar, i.e., requiring strand exchanges. The only significant difference between these two processes is the manner in which the gaps in the sister duplexes are formed, i.e., by excision or by replication bypass (Smith and Sharma, 1987).
For a discussion of excision repair in mammalian cells, see the module Nucleotide Excision Repair in Human Cells.
Summary and Conclusions
It is unfortunate that the importance of recombinational DNA repair is being ignored in many articles on DNA repair. Furthermore, most reviewers make no distinction between the repair events that take place in the two different parts of the chromosome, i.e., the part of the chromosome that was replicated BEFORE UV irradiation, where two DNA duplexes exist, and the part of the chromosome that contains only ONE DNA duplex, and is replicated after UV irradiation. Clearly the problems and the opportunities for DNA repair are quite different in these two regions of the chromosome.
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