Weaver/Molecular Biology

Excision Repair in Eukaryotes

Much of our information about repair mechanisms in humans has come from the study of congenital defects in DNA repair. These repair disorders cause a group of human diseases, including Cockayne syndrome and xeroderma pigmentosum (XP). Most XP patients are thousands of times more likely to develop skin cancer than normal people if they are exposed to the sun. In fact, their skin can become literally freckled with skin cancers. However, if XP patients are kept out of sunlight, they suffer only normal incidence of skin cancer. Even if XP patients are exposed to sunlight, the parts of their skin that are shielded from light have essentially no cancers. These findings underscore the potency of sunlight as a mutating agent. Why are XP patients so extraordinarily sensitive to sunlight? XP cells are defective in NER and therefore cannot repair DNA damage, including pyrimidine dimers, effectively. Thus, the damage persists and ultimately leads to mutations. Since NER is also responsible for repairing chemically-induced DNA damage, we would expect XP patients to have a somewhat higher than average incidence of internal cancers caused by chemical mutagens, and they do. (Notice the underlying assumption here that unrepaired genetic damage can lead to cancer.)

Nucleotide excision repair takes two forms in eukaryotes: It can involve all lesions throughout the genome (global genome NER, or GG-NER), or it can be confined to transcribed regions of the genome (transcription-coupled NER, or TC-NER). The mechanisms of these two forms of NER share many aspects in common, but the method of recognition of the damage differs, as we will see. Let us examine both processes as they occur in humans.

Global Genome NER

What repair steps are defective in XP cells? There are at least eight answers to this question. The problem has been investigated by fusing cells from different patients to see if the fused cells still show the defect. Frequently they do not; instead, the genes from two different patients complement each other. This probably means that a different gene was defective in each patient. So far, seven different complementation groups affecting excision repair have been identified this way. In addition, some patients have a variant form of XP (XP-V) in which excision repair is normal, and the patients’ cells are only slightly more sensitive to ultraviolet light than normal cells are. We will discuss the gene responsible for XP-V later in this chapter. Taken together, these studies suggest that the defect can lie in any of at least eight different genes. Seven of these genes are responsible for excision repair, and they are named XPA-XPG. Most often, the first step in excision repair, incision, or cutting the effective DNA strand, seems to be defective.

The first step in human global genome NER (Figure 8) is the recognition of a distortion in the double helix caused by DNA damage. This is where the first XP protein (XPC) gets involved. XPC, together with another protein called hHR23B, recognizes a lesion in the DNA, binds to it, and causes melting of a small DNA region around the damage. This role in melting DNA is supported by in vitro studies performed in 1997 with templates that contain lesions surrounded by or adjacent to a small "bubble" of melted DNA. These templates do not require XPC, suggesting that this protein’s job had already been performed when the DNA was melted. Also, Sugasawa et al. used DNase footprinting in 1998 to show that XPC binds directly to a site of helix distortion in DNA and causes a change in the DNA’s conformation (presumably a strand separation).

XPA, which has an affinity for damaged DNA, is also involved in an early stage of damage recognition. Since both XPC and XPA can bind to damaged DNA, why do we believe that XPC is the first factor on the scene? Competition studies performed by Sugasawa et al., with different sized templates support this hypothesis. These workers incubated XPC with one damaged template, and all the other factors except XPC with the other damaged template. Then they mixed the two together. Repair began first on the template that was originally incubated with XPC alone, suggesting that XPC binds first to the damaged DNA. Then what is the role of XPA? It can bind to many of the other factors involved in NER, so it appears that it verifies the presence of a DNA lesion, and helps to recruit the other NER factors.

At first, it may seem surprising to learn that two of the other XP genes, XPB and XPD, code for two subunits of the general transcription factor TFIIH, implicating this general transcription factor in NER. However, we now know that these two polypeptides have the DNA helicase activity inherent in TFIIH (Chapter 11). So one role of TFIIH is to enlarge the region of melted DNA around the damage. But TFIIH is required for NER in vitro even with damaged DNAs that have large melted regions, so this protein must have a function beyond providing DNA helicases. The fact that TFIIH interacts with a number of other NER factors suggests that it serves as an organizer of the NER complex.

The melting of the DNA by TFIIH attracts nucleases that nick one strand on either side of the damage, excising a 24-32 nt oligonucleotide that contains the damage. Two excinucleases make the cuts on either side of the damaged DNA. One is the XPG product, which cuts on the 3'-side of the damage. The other is a complex composed of a protein called ERCC1 plus the XPF product, which cuts on the 5'-side. These nucleases are ideally suited for their task: They specifically cut DNA at the junction between double-stranded DNA and the single-stranded DNA created by the TFIIH around the damage. Another protein known as RPA helps position the two excinucleases for proper cleavage. RPA is a single-strand-binding protein that binds preferentially to the undamaged strand across from the lesion. The side of RPA facing toward the 3'-end of this DNA strand binds the ERCC1-XPF complex, while the other side of RPA binds XPG. This automatically puts the two excinucleases on the correct sides of the lesion.

Once the defective DNA is removed, DNA polymerase or fills in the gap, and DNA ligase seals the remaining nick. The role of XPE is not clear yet. Although it is important in vivo, and gives rise to XP symptoms when it is defective, it is not essential in vitro. This makes it more difficult to pin down its function.

Transcription-coupled NER

Transcription-coupled NER uses all of the same factors as does global genome NER, except for XPC. Since XPC appears to be responsible for initial damage recognition and limited DNA melting in global genome NER, what plays these roles in transcription-coupled NER? The answer is RNA polymerase. When RNA polymerase encounters a distortion of the double helix caused by DNA damage, it stalls. This places the bubble of melted DNA, which is created by the polymerase, at the site of the lesion. This somehow signals the other factors to join the complex. From that point on, they behave much as they do in global genome NER, enlarging the melted region, clipping the DNA in two places, and removing the piece of DNA containing the lesion.

Summary: Eukaryotic NER follows two pathways. In global genome NER, a complex composed of XPC and hHR24B initiates repair by binding to a lesion anywhere in the genome and causing a limited amount of DNA melting. This protein apparently recruits XPA and RPA. TFIIH then joins the complex and two of its subunits (XPB and XPD) use their DNA helicase activities to expand the melted region. RPA binds two excinucleases (XPF and XPG) and positions them for cleavage of the DNA strand on either side of the lesion. This releases the damage on a fragment between 24 and 32 nt long. Transcription-coupled NER is very similar to global genome NER, except that RNA polymerase plays the role of XPC in damage sensing and initial DNA melting. In either kind of NER, DNA polymerase or fills in the gap left by the removal of the damaged fragment, and DNA ligase seals the DNA.

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