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FIGURE 9-13 Base excision pathway: the uracil glycosylasc reaction. Uraol glycosylase hydrolyses the glycosîdic bond to release uracil from the DMA backbone to leave an Ai3 site (apuonic or, in this case, apynmtdinic site). AP endo nuclease cuts the DMA backbone at the 5' position of the AP site, leaving a 3'OH, exonudease cuts at the 3' position of the AP site, leading a 5' phosphate The resulting gap is tilled in by ONA polymerase I.

enzyme able to act on the base if it is buried in the helix? The answer to this riddle highlights the remarkable flexibility of DNA. X-ray crys-tallographic studies reveal that the damaged base is flipped out so that it projects away from the double helix, where it sits in the specificity pocket of the glycosylase (Figure 9-14). Interestingly, the double helix

FIGURE 9-14 Structure of a DNA-glycosylase complex. The enzyme is shown in gray and the ONA in purple. Pie dam aged base, in this case oxoG which is shown in red, «s flipped out of the helix and into the catalytic center of the enzyme. (Brunei S.D., Norman DP., and Verdine C.L 2000. Nature 403:859-866. Image prepared wtth Bob Script, WotScript and Raster 3D.)

FIGURE 9-15 oxoG: A repair Oxidation of guanine produces oxoC The modified base can be repaired prior to replication by DMA glycosytee via the base excision pathway if replication occurs before the oxoG is removed resulting in the mistncorporation of an A, then a fail-safe glyccsyfase can remove the A, albv^ng it to be replaced by a C This provides a second opportunity for the DNA glycosylase to remove the modified base.

FIGURE 9-15 oxoG: A repair Oxidation of guanine produces oxoC The modified base can be repaired prior to replication by DMA glycosytee via the base excision pathway if replication occurs before the oxoG is removed resulting in the mistncorporation of an A, then a fail-safe glyccsyfase can remove the A, albv^ng it to be replaced by a C This provides a second opportunity for the DNA glycosylase to remove the modified base.

is able to allow base flipping with only modest distortion to its structure and hence the energetic cost of base flipping may not be great (see Chapter 6 and Figure 6-8). Nevertheless, if is unlikely that glycosylates flip out every base to check for abnormalities as they diffuse along DNA. Thus, the mechanism by which these enzymes scan for damaged bases remains mysterious.

What if a damaged base is not removed by base excision before DNA replication? Does this inevitably mean that the lesion will cause a mutation? In the case of oxoG, which has the tendency to mispair with A, a fail-safe system exists (Figure 9-15). A dedicated glycosylase recognizes oxoG:A base pairs generated by misincorporation of an A opposite an oxoG on the template strand. In this case, however, the glycosylase removes the A. Thus, the repair enzyme recognizes an A opposite an oxoG as a mutation and removes the undamaged but incorrect base.

Another example of a fail-safe system is a glycosylase that removes T opposite a G. Such a T:G mismatch can arise, as we have seen, by spontaneous deamination of 5-methyi cytosine, which occurs frequently in the DNA of vertebrates. Because both T and G are normal bases, how can the cell recognize which is the incorrect base? The glycosylase system assumes, so to speak, that the T in a T:G mismatch arose from deamination of 5-rnethyl-cytosine and selectively removes the T so that it can be replaced with a C.

Nucleotide Excision Repair Enzymes Cleave Damaged DNA on Either Side of the Lesion

Unlike base excision repair, the nucleotide excision repair enzymes do not recognize any particular lesion. Rather, this system works by recognizing distortions to the shape of the double helix, such as those caused by a thymine dimer or by the presence of a bulky chemical ad duct on a base. Such distortions trigger a chain of events that lead to the removal of a short single-stranded segment (or patch) that includes the lesion. This removal creates a single-stranded gap in the DNA. which is filled in by DNA polymerase using the undamaged strand as a template and thereby restoring the original nucleotide sequence.

Nucleotide excision repair in E. coli is largely accomplished by four proteins: UvrA, UvtB, UvrC, and UvrD (Figure 9-16). A complex of UvrA and UvtB scans the DNA, with UvrA being responsible for detecting distortions to the helix. Upon encountering a distortion, UvrA exits the complex and UvrB melts the DNA to create a single-stranded bubble around the lesion, Next, UvrB recruits UvrC, and UvrC creates two incisions: one located eight nucleotides away on the 5' side of the lesion and the other four or five nucleotides away on the 3F side of the lesion. These cleavages create a 12 to 13 residue-long, single-stranded DNA segment, which is made accessible by the action of the DNA helicase UvrD. Finally, DNA polymerase I (Pol 1) and DNA ligase fill in the resulting gap.

The principle of nucleotide excision repair in higher cells is much the same as in E. coli but the machinery for detecting, excising, and repairing the damage is more complicated, involving 25 or more polypeptides. Among these is XPC, which is responsible for detecting distortions to the helix, a function attributed to UvrA in E. coli. As in E. coli, the DNA is opened to create a bubble around the lesion. Formation of the bubble involves the helicase activities of the proteins XPA and XPD (the equivalent to UvrB in E. coli) and the single-strand binding protein RPA. The bubble creates cleavage sites on the 5' side of the lesion for a nuclease known as ERCCl-XPF and on the 3' side for the nuclease XPC (representing the function of UvrC). Tn higher cells, the resulting single-stranded DNA segment is 24 to 32 nucleotides long. As in bacteria, the DNA segment is released to create a gap that is filled in by the action of DNA polymerase and ligase.

As their names imply, the UVR proteins are needed to mend damage from ultraviolet light; mutants of the vvr genes are sensitive to ultraviolet light and lack the capacity to remove thymine-thymine and thymine-cytosine adducts. In fact, these proteins broadly recognize and repair bulky adducts of many kinds. Nucleotide excision repair is important in humans, too. Humans can exhibit a genetic disease called xeroderma pigmentosum, which renders afflicted individuals highly sensitive to sunlight and results in skin lesions, including skin cancer. Seven genes (referred to as XP genes) have been identified in which mutations give rise to xeroderma pigmentosum. These genes correspond to proteins (such as XPA, XPC, XPD, XPF, and XPG, referred to above) in the human pathway for nucleotide excision repair, underscoring the importance of nucleotide excision repair in mending damage from ultraviolet light.

Not only is nucleotide excision repair capable of mending damage throughout the genome, but it is also capable of rescuing RNA polymerase, the progression of which has been arrested by the presence of a lesion in the transcribed (template) strand of a gene. This phenomenon, known as transcription-coupled repair, involves recruitment to the stalled RNA polymerase of nucleotide excision repair proteins (Figure 9-17). The significance of transcription-coupled repair is that it focuses repair enzymes on DNA (genes) being actively transcribed. In cffecl, RNA polymerase serves as another damage-sensing protein in the cell. Central to transcription-coupled repair in eukaiyotes is the general transcription factor TF1IH, As we will see in Chapter 12, TF1IH unwinds the DNA template during the initiation of transcription. Subunits of TFIIII include the DNA helix-opening proteins XPA and XPD discussed above. Thus, TFIIH is responsible for two separate

FIGURE 9-16 Nucleotide excision repair pathway, (a) UvrA and UvrB scan DNA to identify a distortion (b) UvrA leaves the complex', and I ivrfl melts DNA locally around the distortion (c) LMC (orrrs a complex with UvrB and creates ntcks to the 5' side of the lesion and to the 3' side of the lesion, (d) DNA heiicasc UvtO releases the single stranded fragment from the duplex, and DNA Pol I and ligase repair and seal Ihe gap (Source, (parts a-d) Adapted from Zou Y and Van Houten B. 1999. Strand opening by the UvrA3 complex allows dynamic recognition of DNA damage. EMBO Journal 18: 4898, fig 7. Copyright© 1999Oxford University Press. Used with permission)

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