26. Lesions II: Nucleotide Excision Repair (855; 7/28)

Base Excision Repair (BER) fixes relatively subtle chemical damage to individual bases. Nucleotide excision repair (NER) tackles a different class of problem altogether. It removes bulky, helix-distorting lesions--damage large enough to warp the double helix.

These kinds of lesions cause bulges, kinks, or chemical obstructions in the DNA. They can stall DNA polymerases. And very severe lesions can even interfere with the CMG helicase.

There are two ways NER can begin. Here we will only follow the version called global-genome NER, which searches throughout the genome for damaged DNA. (The other is transcription-coupled NER).

Some of these kinds of lesions are caused by ultraviolet light, which can make neighboring DNA bases (especially Cs and Ts) become abnormally bonded to one another. Others are caused by chemicals in tobacco smoke, soot, exhaust, and heavily charred food, which can attach large, flat molecular groups to DNA bases. Neither of these, nor the many other kinds of lesions that NER targets, can be tolerated by the cell.

As with the other repair pathways we've looked at, NER begins with a unique detector protein complex and then transitions into a cut-and-stitch repair pathway. We'll simplify some of the protein names in this chapter. For example, we'll call "XPC-RAD23B" just "XPC."

But unlike BER's approach, with its various and highly specific glycosylase detectors looking for particular chemical changes in the base components of nucleotides, XPC does the opposite. It looks for regions of the genome where the DNA just seems to be behaving abnormally. In other words, BER relies on a collection of specialists while NER takes a more generalist approach.

XPC's method is to repeatedly land on DNA and sample nearby stretches, probing for spots where the helix is loose, distorted, unstable, or difficult to open. This allows one repair system to deal with many chemically unrelated lesions.

Once XPC identifies a potential lesion, it attaches itself to the strand across from it and opens a small bubble in the DNA there. It then recruits a large multi-protein complex (TFIIH) that includes two ATP-powered motor proteins, XPB and XPD.

The other proteins in the TFIIH complex serve to position, regulate and repurpose the two motor proteins between their roles in global-genome NER and transcription-coupled NER.

To verify a lesion, XPB first grips the still-double-stranded DNA a short distance on the 5′ side of the suspected lesion. Using energy from ATP, it partially unwinds the DNA between itself and XPC, enlarging the bubble and exposing even more of the strand that may contain a lesion.

XPD then grips the strand suspected of containing the lesion and uses its own motor to actively pull that strand through a narrow channel in the protein. As the DNA passes through, XPD is inspecting it. Normal DNA can pass, but a bulky damaged nucleotide can obstruct the channel and cause XPD to stop.

This is another example of the general idea of steric hinderance and so similar to how the MCM component of the CMG helicase separates the two strands of the double helix.

If the DNA being pulled through doesn't get through or slows down, then this suggests that a lesion is, in fact, present and the site is an appropriate target for NER. If the suspicious region passes through XPD without being slowed, NER is less likely to proceed.

Once the lesion has been verified, two cutting enzymes--XPF and XPG--are recruited and act on the damaged strand. XPF cuts on the 5′ side, usually about 20 nucleotides in front of the lesion. XPG cuts much closer on the 3′ side, generally about five nucleotides beyond it. These cuts release a short piece of single-stranded DNA—roughly 25–30 nucleotides long—that contains the lesion. That short pice of DNA is degraded by the cell and the lesion is gone.

At the same time, RPA proteins arrive. These RPAs coat and stabilize the intact complementary strand, while at the same time keeping it available as the template for repair.

With the damaged segment now removed, NER reverts to the familiar cut-and-patch process involving now-familiar enzymes. A DNA polymerase—usually the leading or lagging strand DNA polymerase, but sometimes another I haven't mentioned yet—fills the gap by copying the intact complementary strand. PCNA helps hold the polymerase on the DNA. Finally, a DNA ligase seals the remaining nick.

BER and NER are, again, both variations of cut-and-patch repair pathways. But while BER targets chemistry (single altered bases) NER targets structure (lesions that warp the helix). And while BER performs microsurgery on one base, NER removes the entire patch of DNA surrounding the problem.

Also note that neither pathway simply reads damaged DNA while it remains comfortably inside the helix. Both disturb the helix and expose bases so that the damage can be evaluated more closely.

But what happens when a moving replisome collides with a lesion that hasn't yet been repaired by BER or NER? When this occurs, DNA synthesis may stall. If the obstacle cannot be bypassed or repaired, the replication fork can become unstable and, in severe cases, collapse.

We turn next to what happens when un-repaired DNA lesions meet the moving replication machinery.