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functions: ita strand-separating helicases melt the DNA around a lesion during nucleotide excision repair (including transcription-coupled repair) and also help to open the DNA template during the process of gene transcription. Systems for coupling repair to transcription also exist in prokaryotes.

Recombination Repairs DNA Breaks by Retrieving Sequence Information from Undamaged DNA

Excision repair uses the undamaged DNA strand as a template to replace a damaged segment of DNA on the other strand, flow do cells repair double-strand breaks in DNA in which both strands of the duplex are broken? This is accomplished by the double-strand break (DSB) repair pathway, which retrieves sequence information from the sister chromosome. Because of its central role in general, homologous recombination as well as in repair, the DSB-repair pathway is an important topic in its own right, which we shall consider in detail in Chapter 10.

DNA recombination also helps to repair errors in DNA replication. Consider a replication fork that encounters a lesion in DNA (such as a thymine dimer) that has not been corrected hv nucleotide excision repair. The DNA polymerase will sometimes stall attempting to replicate

nucleotide excision repair proteins transcription nucleotide excision repair proteins over the lesion. Although the template strand cannot be used, the sequence information can be retrieved from the Other daughter molecule of the replication fork by recombination (see Chapter 10). Once this »¡combinational repair is complete, the nucleotide excision system has another opportunity to repair the thymine dimer. Indeed, mutants defective in recombination are known to be sensitive to ultraviolet light. Consider also the situation in which the replication fork encounters a nick in the DNA template. Passage of the fork over the nick will create a DNA break, repair of which can only be accomplished by the double-strand break repair pathway. Although we generally consider recombination as an evolutionary device to explore new combinations of sequences, it may be that its original function was to repair damage in DNA.

The DSB-repair pathway can only operate when the sister of the broken chromosome is present in the cell. What happens when a chromosome breaks early in the cell cyclc, before a sister has been generated by DNA replication? Under these circumstances, a fail-safe system comes into play known as nonhomologous end joining (NIIEJ), As its names implies, NHEJ does no! involve homologous recombination. Instead, the two ends of the broken DNA are directly joined to each other by misalignment between single strands protruding from Lite broken ends. This misalignment is believed to occur by pairing between tiny stretches (as short as one base pair) of complementary bases (serendipitous microhomologies). Single-stranded tails are removed by nucleases and gaps are filled in by DNA polymerase. NHFJ is mediated by Ku, a member of a widely-conserved family of proteins found in bacteria, yeast and hurnans. Ku proteins align the ends of broken chromosomes, protect them from nucleuses, and recruit other repair proteins. Ku-mediated NHPJ is an inefficient process (allowing survival of only one in a thousand yeast cells in which a chromosome break has been introduced) and leads to the formation of deletions ranging in size from a few base pairs to several kilobases at the site at which the chromosome breakage originally occured.

Translesion DNA Synthesis Enables Replication to Proceed across DNA Damage in the examples we have considered so far, damage to the DNA is mended by excision followed by resynthesis using an undamaged template. But such repair systems do not operate with complete efficiency and sometimes a replicating DNA polymerase encounters a lesion, such as a pyrimidine dimer or an apurinic site, that has not been repaired, Because such lesions are obstacles to progression of the DNA polymerase, the replication machinery must attempt to copy across the lesion or be forced to cease replicating. Even if cells cannot repair these lesions, there is a fail-safe mechanism that allows the replication machinery to bypass these sites of damage. This mechanism is known as translesion synthesis. Although this mechanism is. as we shall see, highly error-prone and thus likely to introduce mutations, translesion synthesis spares the cell the worse fate of an incompletely replicated chromosome.

Translesion synthesis is catalvzed by a specialized class of DNA polymerases that synthesize DNA directly across Lhe site of (he damage (Figure 9-18). Transits ton synthesis in B. coli is carried out by a complex of the proteins UrnuC and UnitiD'. UrnuC is a member of a distinct family of DNA polymerases found in many organisms known as Lhe Y-family of DNA polymerases (Figure 9-19 and Box 9-3, The Y-Farnily of DNA Polymerases).

FICURt 9-18 Translesion DNA synthesis.

Upon encountering a lesion in the template during replication. DNA polymerase III with its sliding clamp dissociates from the DNA and is replaced by the irarrslesion DNA polymerase, which extends DNA synthesis across the thymine dimer on the template (upper) strand The translesion polymerase is then replaced by the DNA polymerase 111, (Source: R. Woodgate.)

DNA^polymerase III

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