The Fix-It Crew I

Life can't exist without the high fidelity transmission of genetic information from parent cell to daughter cell. DNA polymerases plays a big role in accomplishing this. DNA polymerases delta and epsilon only make an error (that is, attach a non-complementary nucleotide to a growing chain) once every 100,000 to 1 million nucleotide additions. But this is too many. They'll need to be fixed via a back-up system that we'll soon be discussing.

In addition, the human genome--even in healthy cells--is under constant assault from endogenous (internal to the cell) and exogenous (external to the cell, or environmental) chemicals and other agents that alter its chemistry, break its backbone, and distort its helical structure. We'll refer to such alterations as "lesions" to distinguish them from errors. They'll need to be fixed, too.

In this and the next three posts, we'll learn how cells handle these issues. The umbrella term for the cell's response to these kinds of threats is DNA repair. DNA repair consists of an array of specialized pathways that, together, correct all replication errors, remove lesions both large and small, and rebuild damaged regions of the genome.

Errors and Lesions

Let's spend a moment on errors and lesions. The most common errors are substitutions. These are pretty straightforward: they occur when a DNA polymerase adds a non-complementary nucleotide to a growing chain. For example, given an "A" on the template strand, the DNA polymerase might accidentally pair it with a "C" instead of the correct "T."

In addition to substitutions, sometimes the DNA polymerase slips, and either fails to add one or more nucleotides (creating a deletion) or adds one or more extra nucleotides (creating an insertion). Insertions and deletions are rarer than substitutions, but they do occur. The cell needs the ability to fix all these kinds of errors.

Regarding lesions, most (90-99%) are the result of endogenous rather than exogenous insults. These are caused by the natural byproducts of cell metabolism and by the cell's aqueous environment. Many metabolic byproducts are reactive and chemically alter DNA, especially the base component. They can also cause complete removal of a base from a nucleotide and single- and double-stranded breaks. Lesions are not rare. Even a healthy human cell accumulates tens of thousands of endogenous DNA lesions every day.

Causes of the rarer exogenous DNA lesions (1-10% of the total) include UV radiation from the sun and reactive chemicals from environmental pollutants, industrial chemicals, dietary products, cigarette smoke and air pollution. UV exposure is responsible for a common kind of lesion that we'll be discussing later: an unwanted bond that forms between neighboring bases. Exogenous insults also cause some other very serious lesions that we'll discuss later.

Why DNA Repair?

Now, let's pause for a moment. You might be wondering why we're focusing attention to DNA repair given that our main topic is DNA replication. The two, it turns out, are largely integrated with each other. First, as I've mentioned, replication is the primary generator of substitution errors and small insertions and deletions. Repairing these errors takes place inside the replications fork immediately after nucleotide addition. So error repair is highly coordinated with DNA replication.

In addition, lesions are like DNA replication roadblocks. They interfere with DNA polymerases and, in some cases, CMG helicases as they translocate along the DNA. A lesion can halt a replisome in its tracks. Thus, DNA replication is dependent on DNA repair. So we can't ignore it. Nor do we want to. It's one of the most amazing activities of cells.

I'm going to be categorizing DNA repair systems as follows:

1. Error correction systems include Polymerase Proofreading and Mismatch Repair (MMR). MMR handles the errors that get past proofreading, fixing them immediately after synthesis. Error correction also fixes loops in DNA caused by a small insertions and deletions of nucleotides made by the polymerase.

2. Damage processing systems focus on lesions rather than errors. Some act, like MMR, at or very near the replications forks. These include Ribonucleotide Excision Repair (RER), some aspects of Base Excision Repair (BER), and Translesion Synthesis (TLS). We'll also briefly discuss Nucleotide Excision Repair (NER) even though it isn't associated with replication.

3. Fork recovery and restart systems. These address the most serious lesions. Repair pathways include Homologous Recombination (HR) and the Fanconi Anemia (FA) Pathway, both of which are sometimes preceded by fork reversal. These are complex, multi-step pathways, but essential for cell survival.

I'l cover the first category--error correction systems--in this post. I'll use the next two post to cover categories two and three.

DNA Polymerase Proofreading

Here's a quick refresher on DNA polymerases delta and epsilon's on-board error correction functions based on their 3' to 5' exonuclease activities.*

In the rare event that DNA polymerase delta or epsilon attaches a non-complementary nucleotide to a nascent DNA chain, the enzyme senses this (based on a conformation change caused by the mispairing) and pauses. It then transfers the 3' end of the nascent strand to another site on the enzyme that has 3' to 5' exonuclease activity. The exonuclease removes about 2-5 nucleotides, including, of course, the mismatched one. The 3' end is then returned to the polymerase active site where the few nucleotides just removed are added back. Then the replisome continues on its way.

How effective is DNA polymerase proofreading? As I just mentioned, the baseline mismatch rate (that is, before proofreading) of a replicative polymerase is 1 error every 100,000-1 million nucleotides. Polymerase proofreading improves that 100- to 1000-fold. That equates to a mismatch, or error, rate after polymerase proofreading of 1 error every 10 million to 100 million nucleotides! But even this isn't good enough. Thus, mismatch repair (MMR).

Mismatch repair (MMR)

Mismatch repair (MMR) corrects errors that DNA polymerase proofreading fails to fix. It occurs within the replication fork, its initial detector enzyme being physically linked to the PCNA sliding clamp.

MMR as well as several of the lesion-fixing pathways use a similar five step process that's referred to as "cut-and-patch" repair to fix errors and small- to medium-sized lesions. We're going to see variations of this approach in this and the next two posts--in mismatch repair (MMR), ribonucleotide excision repair (RER), base excision repair (BER) and nucleotide excision repair (NER). So let's review the steps here. Then I'll focus on MMR.

In cut-and-patch repair, the following generic steps occur in order:

1) an error or lesion is identified and marked by a detector protein

2) an enzyme makes a single-stranded nick in the DNA close to the error or lesion

3) another enzyme starts at the nick and chews back the DNA, removing the error or lesion

4) a DNA polymerase uses the "good strand" as the template to fill-in the nucleotides that were chewed back

5) a DNA ligase seals the gap, creating a continuous strand

Now let's look at MMR specifically to make these steps more tangible. Generic step 1 is identifying and marking the error or legion. In MMR three specific proteins detect errors. MMR's core detector protein is MSH2. It pairs with one of two other proteins--MSH6 or MSH3--to perform its detection function.

When MSH2 pairs with MSH6 the heterodimer is called MutS-alpha. It identifies single base mismatches and small loop structures caused by insertions and deletions of 1-2 nucleotides. These represent about 85-90% of all errors. When MSH2 pairs with MSH3 the heterodimer is called MutS-beta. It detects larger (2-13 nucleotides) insertions and deletions, amounting to 10-15% of all DNA polymerase errors.

MutS-alpha and MutS-beta tether to the back face of PCNA. There, they continuously scan the DNA as it's synthesized, looking for DBNA polymerase errors. Any mismatch or small loop distorts the DNA, creating a small kink. The kink, in turn, creates a small gap between the strands of the double helix. Once the MutS protein complex detects the kink, it inserts a specific amino acid into the gap to stabilize the kink so that it clearly flags the error.

When this amino acid is inserted into the double helix gap, it also causes conformation changes in MutS-alpha and MutS-beta that closes them around the DNA, turning them into sliding clamps (unrelated to PCNA) near the mismatch or loop. The MutS-alpha sliding clamp marks the mismatch and provides a docking station for the next actor in the pathway, MutL-alpha.

Mismatch Repair (MMR) pathway. MutS-alpha and -beta identify and mark the error and then recruit MutL-alpha. MutL-alpha makes an incision in the error strand using its endonuclease activity and then recruits other proteins needed to finish the job. These include EXO1 exonuclease to remove the mismatched nucleotide(s), RPAs to protect single-stranded DNA, DNA polymerase delta or epsilon to fill in the nucleotides removed by EXO1, and DNA ligase I to connect the strands.

Like MutS-alpha, MutL-alpha is a heterodimer that performs two roles. It makes a single-stranded incision in the DNA strand with the mismatch. Recall that this is the second step of our "cut-and-patch" repair model. It then recruits the next set of required proteins including EXO1, RPAs, DNA polymerase delta and DNA ligase I. Thus, MutL-alpha performs step 2 (the incision) then it orchestrates the transition to steps 3 and 4 (excision and fill-in).

MutL-alpha makes an incision in the "bad" strand somewhere 5' of the mismatch. Let's pause. MutL-alpha's job is to cut the strand containing the error. But how does MutL-alpha "known" which of the two strands contains the error? One strand contains the correct nucleotide and the other the incorrect one. Which is which?

MutL-alpha uses two kinds of clues to determine which strand to cut. On the lagging strand, any gaps between Okazaki fragments that haven't yet been filled by DNA ligase identify the new strand, which is the strand with the error. Cut the strand with the gaps!

Because the leading strand contains no nicks in its newly synthesized strand, MutL-alpha needs a different clue: the orientation of the PCNA sliding clamp. The PCNA disk has a front and a back face. Since DNA polymerase only synthesizes 5' to 3', the strand exiting the PCNA's back face with its 5' end is the strand to nick!

The incision made, we're now at the third step of "cut-and-patch" repair: chewing back the DNA containing the error. In MMR, EXO1 exonuclease (a 5' to 3' exonuclease) chews the strand with the error starting at the incision. The nick might be close to the error or it might be several hundred or even a thousand nucleotides 5' of the mismatch.

As EXO1 chews back the "bad" strand, how will it "know" when it has removed the error? Here's how. Given that the MutS-alpha sliding clamp is flagging the error, by the time EXO1 runs into it, the error will have been removed! The MutS sliding clamp serves as a molecular stop signal, or roadblock, for EXO1.

Of course, as EXO1 is chewing back the strand containing the error, the complementary strand is being exposed to destructive nucleases. By now we know what that means. RPAs (single-stranded binding proteins) will be needed. They arrive and coat the complementary single strand to protect it.

DNA polymerase delta arrived next to perform step four of our MMR "cut-and-stitch" repair. Starting at the nick, and with the mismatch "bad" strand now absent due to the action of EXO1, DNA polymerase delta synthesizes a new strand. It continues until it reaches the 5' end of the strand right in front of it. At that point, it stops. DNA polymerases can't connect two abutting strands. That job goes to DNA ligase 1, which performs the fifth and last step in "cut-and-stitch" repair: it ligates the two strands.

The error--be it a substitution, an insertion, or a deletion--has been corrected. It typically takes human cells between several minutes and 30 minutes to complete the MMR pathway and repair an error. It does this simultaneous with replication fork movement. The first seconds are critical, however, because only then are there clues--in the form of lagging strand Okazaki fragment gaps and leading strand PCNA orientation--as to which strand or nucleotide to fix.

• We refer to the different functions that an enzyme can perform as that enzyme's "activities." So DNA polymerase epsilon, for example, has two activities: a DNA polymerization activity and a 3' to 5' exonuclease activity.