25. Lesions I: Base Excision Repair (1,116; 7/26)

In addition to DNA polymerase errors, the human genome--even in a perfectly healthy cell--is under intense assault. The replication errors we just discussed are mistakes made while copying DNA. Lesions are different. They're molecular injuries inflicted throughout the cell cycle on otherwise accurately copied DNA.

Lesion-causing threats come from within and from without. Chemicals generated by normal cell metabolism react with DNA. 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.

In this chapter, we’ll first get more specific about what we mean by a lesion. Then we will focus on the first of two repair systems that correct the majority of DNA lesions: base excision repair (BER) and nucleotide excision repair (NER).

Recall the structure of a nucleotide. Each has a backbone component and a base component. The backbones of many connected nucleotides form one of the rails of the double helix ladder. The bases provide the genetic information.

Sometimes in the cell, only the base component of a nucleotide is affected by a lesion. When this occurs, BER performs the repair. At other times, multiple bases or a whole nucleotide is affected--often by bulky, helix-distorting insults. In those cases, NER performs the repair. The cell employs the appropriate pathway given the type or severity of the lesion.

Like MMR and RER, the BER and NER pathways are variations of cut-and-stitch repair. But unlike MMR and RER, which are closely associated with DNA replication and act mainly during and shortly after S phase when DNA is being actively synthesized, BER and NER repair DNA damage during all phases of the cell cycle. They're always at work.

To be technically accurate, BER is really two related pathways: short-patch BER and long-patch BER. Short-patch BER repairs a single damaged base on a nucleotide. Long-patch BER handles larger base-related repair challenges. I'll only cover short-patch BER. Conceptually, little will be missed by only covering only one of them.

Let’s start out by looking at the kinds of lesions that these pathways address.

DNA lesions

Most DNA lesions--upwards of 90 percent--arise from internal chemical causes rather than environmental ones. Normal cellular metabolism generates reactive molecules that chemically modify DNA bases. Water, ubiquitous in the cell, also slowly reacts with DNA.

These processes can add, remove, or alter chemical groups specifically on bases. Sometimes they eliminate a base altogether. Lesions caused by the chemical milieu of the cell are not rare. A healthy human cell accumulates tens of thousands of internally-caused DNA lesions every day.

Environmental causes account for less than ten percent of lesions. But they effect more dramatic damage. For example, 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, a C and/or a T).

In addition, reactive chemicals from pollutants, cigarette smoke, industrial chemicals, and diet can attach bulky chemical adducts to DNA. External insults may be less frequent, but they produce some of the most disruptive lesions.

The exact number is difficult to determine, but a human cell probably experiences tens of thousands of DNA lesions each day. Many are small alterations that are handled primarily by BER. Spontaneous loss of purine bases (A and G bases) alone is estimated to produce roughly 10,000 abasic sites per cell per day. The number of bulky lesions handled by NER is much more dependent on environmental exposure, particularly ultraviolet light and reactive chemicals.

Given the large number of lesions impacting the genome , we need to start thinking of the genome as chemically dynamic--constantly altered and constantly repaired. BER especially should be thought of as a continuous molecular maintenance system.

Base Excision Repair (BER)

Base excision repair (BER) targets damage to the base portion of a nucleotide--the A’s, T’s, G’s, and C’s themselves. If left unrepaired, altered bases can mis-pair during replication, creating mutations. They can also stall DNA polymerases and therefore replication.

Base lesions include many different types of relatively small chemical changes--a missing chemical group, an added one, or a subtle structural alteration. BER is perfectly suited to fix all of these. The pathway begins with an entire family of detector proteins called DNA glycosylases.

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

These various glycosylases patrol DNA constantly. They move along the double helix, literally bending it as they go, testing for local instability--the kind that would suggest a chemically altered base. If one of the glycosylases 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 the damage is real--the glycosylase cleaves the bond between the base and the backbone component of the nucleotide and removes the base. What remains is an abasic site: a nucleotide in the backbone missing its base. This is step one.

Next, a specific endonuclease--different than the one that takes part in MRR--recognizes abasic sites and cuts the DNA backbone just in front of it (technically, just 5′ of it), creating a single-strand nick.

Now a new specialized DNA polymerase enters the story. We'll call this the BER DNA polymerase since its primary, if not exclusive, role is in BER. This special DNA polymerase performs two jobs. First, it removes the remaining backbone remnant of the abasic nucleotide creating a one nucleotide gaps. Then it inserts the correct single nucleotide into that space using the parental strand as the template. At this point, only a nick remains.

A DNA ligase--once again, different from the DNA ligase involved in MMR and RER--now seals the remaining nick. It usually works in partnership with a scaffolding protein called XRCC1.

XRCC1 is not an enzyme. It is a flexible organizing molecule that holds the DNA ligase near the DNA polymerase and other repair proteins, allowing the fragile intermediates of BER to be passed efficiently from one enzyme to the next.

Recall that replication-coupled pathways such as MMR and RER use a different organizing system: the PCNA sliding clamp, which recruits the DNA ligase and other enzymes to the newly synthesized DNA. The BER DNA polymerase, however, works without PCNA. It doesn't need to. It doesn't remain on the DNA for a long period of time. It only needs to insert one new nucleotide.

A single damaged base has now been detected, removed, replaced, and the DNA has been re-sealed--all without replacing surrounding DNA. That's short-patch BER.

Next we look at nucleotide excision repair (NER), which targets much larger and more disruptive lesions.