25. Lesions I: BER and NER (1,557) DONE

In addition to DNA polymerase errors, the human genome — even in a perfectly healthy cell — is under intense assault.

The threats come from within and without. Endogenous chemicals generated by normal metabolism react with DNA. Exogenous agents from the environment — sunlight, pollutants, cigarette smoke, industrial chemicals — do the same. Together they alter DNA’s chemistry, nick its backbone, and distort its double helical structure.

Replication errors are mistakes made while copying DNA. Lesions are different. They are injuries inflicted on DNA itself.

In this chapter, we’ll first clarify what we mean by a lesion. Some lesions are modest — a small chemical modification to a single base. Others are dramatic — for example, a DNA–protein crosslink (DPC), in which a large protein becomes strongly attached to DNA, effectively gluing itself to the genome.

Most of our attention in this chapter will focus on the two repair systems responsible for correcting the majority of DNA lesions: base excision repair (BER) and nucleotide excision repair (NER).

Before diving in, one important distinction.

BER is really two related pathways: short-patch BER and long-patch BER. Short-patch BER, repairs a single damaged base on a nucleotide. It operates throughout the genome and throughout the cell cycle. It is not tied to replication.

Long-patch BER handles somewhat larger repair challenges and generally operates near the replication fork, where it borrows heavily from the enzymatic machinery used during Okazaki fragment processing.

I'll cover only on short-patch BER. Both employ variations of cut-and-stitch repair mechanisms. Not much conceptually will be missed by only describing one of them.

NER, unlike BER, removes very bulky, helix-distorting lesions. Like short-patch BER, it is performed well beyond the replisome and throughout the cell cycle.

In short: some repair pathways work hand-in-hand with replication. Others--including the two we'll discuss here--patrol the genome continuously, independent of it.

Now let’s look more closely at the kinds of lesions these pathways address.

DNA lesions

Chemical alterations to DNA are called lesions--a term we use to distinguish them from DNA polymerase “errors.” A lesion is any change to DNA that disrupts its normal chemical structure.

This distinction is important. With errors--substitutions, insertions, and deletions--the nucleotides themselves remain chemically intact. They’re simply misplaced or mismatched. Even a ribonucleotide inserted into DNA is chemically correct--just in the wrong place.

Lesions are different. They're injuries to the molecule itself.

Most DNA lesions--somewhere between 90 and 99 percent--arise from endogenous causes rather than environmental ones. They are the unavoidable byproducts of life inside the cell.

Normal cellular metabolism generates reactive molecules that chemically modify DNA bases. Water, ubiquitous in the cell, slowly reacts with DNA as well. These processes can add, remove, or alter chemical groups on bases--sometimes eliminating a base altogether.

Lesions--especially endogenous ones--are not rare. Even a healthy human cell accumulates tens of thousands of endogenous DNA lesions every day.

Environmental causes account for a smaller fraction--perhaps 1 to 10 percent--but they often produce more dramatic damage. Ultraviolet (UV) radiation from sunlight can induce abnormal bonds between neighboring bases, including a common lesion we’ll examine shortly: an unwanted strong bond between adjacent pyrimidine bases (that is, C and/or T).

Reactive chemicals from pollutants, cigarette smoke, industrial compounds, and even dietary sources can attach bulky chemical adducts to DNA. Exogenous insults may be less frequent, but they produce some of the most structurally disruptive lesions.

The genome, in other words, is chemically dynamic. It is constantly being altered--and constantly being repaired. And to perform those repairs, the cell deploys different repair strategies depending on the size and structural impact of the damage.

Base Excision Repair (BER)

For perspective, recall RER. There, the problem was the sugar portion of the nucleotide — a ribose instead of a deoxyribose. RER fixed the sugar.

Base excision repair (BER) has a different target. It repairs damage to the base portion of the nucleotide — the A’s, T’s, G’s, and C’s themselves.

Base damage is very common. A single human cell experiences tens of thousands of base lesion events per day.

If left unrepaired, altered bases can mis-pair during replication, creating mutations. They can also stall DNA polymerases. Given how frequently base damage occurs, the cell must constantly monitor and repair its DNA bases.

Fortunately, most base lesions involve relatively small chemical changes — a missing chemical group, an added one, or a subtle structural alteration. BER is perfectly suited to fix these.

Short-patch BER

Short-patch BER operates throughout the genome and throughout the cell cycle. It's not tied to replication. It's a continuous molecular maintenance system. But it is a classic example of cut-and-patch repair.

It begins with an entire family of detector proteins called DNA glycosylases.

Because many different types of base damage can occur, the cell uses multiple glycosylase proteins — roughly 10 to 12 distinct enzymes--each specialized to recognize a specific type of altered base.

These glycosylases patrol DNA constantly. They move along the double helix, subtly bending it as they go, testing for local instability--the kind that would suggest a chemically altered base.

If a glycosylase detects instability, it flips the suspect base out of the helix and into a pocket within the enzyme. If the base fits that pocket — meaning it truly is damaged — the glycosylase cleaves the bond between the base and the sugar, removing the base.

What remains is an abasic site: a nucleotide backbone missing its base.

This is step one.

Next, APE1 (AP endonuclease 1) recognizes the abasic site and cuts the DNA backbone just 5′ of it, creating a single-strand nick.

Now a new polymerase enters the story: DNA polymerase-beta.

It performs two jobs. First, it removes the remaining sugar-phosphate remnant of the abasic nucleotide. Second, it inserts the correct nucleotide using the undamaged strand as the template. At this point, only a nick remains.

A scaffold protein, XRCC1, coordinates the final step by recruiting DNA ligase III, which seals the nick and restores strand continuity.

One damaged base has been detected, removed, replaced, and sealed — all without replacing surrounding DNA. That is short-patch BER.

Nucleotide Excision Repair (NER)

If base excision repair fixes subtle chemical damage to individual bases, nucleotide excision repair (NER) tackles a different class of problem altogether. NER removes bulky, helix-distorting lesions — damage large enough to warp the DNA double helix.

These lesions don’t just alter chemistry. They alter structure.

Two classic examples:

• A pyrimidine dimer, formed when ultraviolet (UV) radiation causes two adjacent cytosine and/or thymine bases to become strongly linked.

• A bulky chemical adduct, or attachment, such as those formed by carcinogens in cigarette smoke.

  
  

Both distort the helix. Both can stall DNA polymerases. Some are large enough to impede even the CMG helicase.

Unlike BER, which removes a single base, NER removes an entire stretch of nucleotides — typically 24–32 bases — containing the lesion. It's a more dramatic repair strategy for more dramatic damage.

Detecting helix distortion

NER begins with a protein complex called XPC–RAD23B.

Unlike BER glycosylases, which look for specific chemical changes, XPC–RAD23B is a detector protein that searches for structural distortions. It patrols the genome, probing for irregularities in the double helix itself.

When it finds a distorted region, it inserts a structural “arm” between the strands and flips out the bases opposite the lesion, creating a small unwound bubble. Importantly, XPC–RAD23B binds to the undamaged strand, marking the opposite strand as the one containing the lesion. This orientation will matter.

Step 2. Verifying and expanding the bubble

Once distortion is detected, XPC–RAD23B recruits a large multiprotein complex called TFIIH.

TFIIH has two helicase subunits: XPB and XPD. Together, they enlarge the bubble around the lesion.

XPB initiates local opening, unwinding approximately 20–30 nucleotides of DNA. XPD then moves along the exposed single strand, scanning it for an obstruction.

If XPD progresses smoothly, the distortion may not represent a lesion serious enough to warrant NER. The process aborts. But if XPD stalls, that stall confirms the presence of a bona fide lesion. NER proceeds.

This verification step prevents unnecessary excision of undamaged DNA.

Dual incisions

Once verified, two endonucleases arrive.

• XPF–ERCC1 cuts the damaged strand on the 5′ side, roughly 5 nucleotides upstream of the lesion.

• XPG cuts on the 3′ side, roughly 25–30 nucleotides downstream.

These coordinated cuts release a short single-stranded DNA segment — about 24–32 nucleotides long — containing the lesion.

TFIIH helps displace this fragment. RPA proteins stabilize the undamaged complementary strand to prevent re-annealing. The excised fragment is degraded. The lesion is gone.

Fill and Seal

With the damaged segment removed, NER reverts to a familiar cut-and-patch process.

DNA polymerase delta or epsilon, held in place by PCNA, synthesizes new DNA using the intact strand as the template.

Finally, DNA ligase I seals the remaining nick. The double helix is restored.

The big picture

As we've seen, BER and NER are both variations of cut-and-patch repair pathways. But while BER targets chemistry--single altered bases, NER targets structure--lesions that warp the helix. Both target lesions in the genome at large and they do it throughout the cell cycle.

But what happens when a lesion is encountered during replication itself — when a moving replisome collides with damage that hasn't yet been repaired?

When this occurs, the replication fork can stall. And then it can collapse. Alternatively, it can be stabilized and protected. We turn next to what happens when DNA damage meets the moving replication machinery.