Polymerase Errors II

As I mentioned in the last post, DNA polymerases sometimes insert a ribonucleotide (an RNA monomer) into a growing chain instead of a deoxyribonucleotide (a DNA monomer). When that happens, the bases do pair correctly. But the extra small chemical group that differentiates a deoxyribonucleotide from a ribonucleotides can still causes problems like strand breakage.

DNA vs RNA. The sugar portion in a ribonucleotide is a ribose whereas in a deoxyribonucleotide it is a deoxyribose. Green numbering indicates carbon atoms in the molecule. Note the small difference at carbon position 2'. A small chemical group (a hydroxyl) is missing on the deoxyribose. Also note that it is this carbon numbering that determines the orientation of a DNA strand (5' vs. 3')

Ribonucleotide mis-incorporations occur frequently--much more often than deoxyribonucleotide mis-incorporations. There are two reasons. First, DNA polymerase's ability to discriminate ribonucleotides from deoxyribonucleotides isn't perfect; the two molecules differ only by one small chemical attachment (see figure). The second reason is that ribonucleotides are present in the nucleoplasm at 20-100 times higher concentration than deoxyribonucleotides. These are the raw materials of mRNAs, which the cell manufactures constantly and in large numbers to build the proteins it needs. Since they're ubiquitous in the nucleus, they're mis-incorporated much more often than non-complementary deoxyribonucleotides.

How frequently are ribonucleotides misincorporated? I stated earlier that the deoxyribonucleotide mismatch error rate after polymerase proofreading (but before MMR) was one error every 10 million to 100 million nucleotides. In sharp contrast, the ribonucleotide insertion rate is once every 1,000 to 5,000 nucleotides! That's an enormous difference. The cell is constantly correcting mis-incorporated ribonucleotides!

Ribonucleotide Excision Repair (RER)

Like MMR, RER is an example of a "cut and patch" repair system. To review, in such a system, first a lesion is detected. Then a single strand incision is made 5' of the lesion (that's in front of the lesion from the polymerase's perspective). Then the DNA near the removed lesion is replaced using information from the "good" strand. Finally, the two pieces of now-repaired single-stranded DNA are reconnected to restore the continuity of the strand.

RER uses many of the same proteins that take part in the removal of the RNA portions of the primers used to make Okazaki fragments in lagging strand synthesis. That makes sense. In both cases, the job is to remove one or more ribonucleotides. Given that most of the Okazaki fragment processing proteins are replisome-linked, it's also no surprise that RER, Okazaki fragment processing, occurs right after DNA synthesis just behind the replisome.

RER starts with the protein RNase H2, which has two jobs. It uses its detector activity to suss out ribonucleotides that have been incorporated into the growing chain. It then uses its endonuclease activity to make an incision immediately 5' of that ribonucleotide.

Recall that RNase H2 is also the enzyme that made endonucleolytic cuts to remove the first 10 or so ribonucleotides at the 5' end of Okazaki fragment primers. So in RER, RNase H2 perfoms the first step in "cut-and-patch" repair (identifying the lesion) as well as the second step (making a single strand incision near the lesion).

Once the incision is made, the PCNA sliding clamp recruits DNA polymerase delta to the nick. In a classic "cut-and-patch"repair process, the next step would be to chew up the strand with the mis-incorporated ribonucleotide. But RER is more like Okazaki fragment processing: DNA polymerase delta synthesizes off the DNA 3' end of the incision, using the complementary "good" strand as the template. As it synthesizes 5' to 3', it displaces the strand containing the mis-incorporated ribonucleotide and several deoxyribonucleotides that follow, creating a flap.

The flap will end up being 1-10 nucleotides long and will be cleaved off by FEN1 (Flap Endonuclease I), the same enzyme that performed the same task in Okazaki fragment processing. Once FEN1 cleaves off the flap, only one more job remains: connecting, or ligating, the DNA. That falls to the enzyme that joined abutting Okazaki fragments: DNA ligase I. Once strand continuity is restored, the repair is complete.

Ribonucleotide Excision Repair (RER). The pathway includes several of the enzymes used in Okazaki fragment processing including RNase H2, DNA polymerase delta, the PCNA sliding clamp, FEN1 and DNA ligase I.

As I mentioned, RER is tightly coupled to DNA replication. All the enzymes involved in RER tether to the back face of the PCNA sliding clamp including RNase H2, DNA polymerase delta, FEN1, and DNA ligase I. Because it is so closely linked with DNA replication, and because ribonucleotide mis-incorporations are so common, RER can be thought of as an obligatory quality control step in a DNA manufacturing process. RER succeeds in correcting 99-99.9% of mis-incorporated ribonucleotides during S phase.

A Complete Error Correction Solution

As we've seen over the past two posts, the cell has several repair pathways that target DNA polymerase-caused errors. The first is the DNA polymerase itself with its proofreading function. Much more often than not, the enzyme senses a mis-incorporated deoxyribonucleotide. Once it does, it uses its 3' to 5' exonuclease activity to fix it. The number of mis-incorporation errors left after polymerase proofreading is about one error every 10 million-100 million nucleotides.

Those deoxyribonucleotide mis-incorporation errors that get past DNA polymerase proofreading will be sensed by MutS-alpha and repaired within the replisome via MMR, which uses a "cut-and-patch" approach. The number of mis-incorporation errors remaining after MMR is about one error every 1 billion to 10 billion nucleotides. Per cell division, that equates to anywhere from zero to a handful (5-6) errors among the 6.2 billion nucleotides of DNA in the cell's two genomes.

Finally, the most common kind of error caused by DNA polymerases is ribonucleotide insertions, which occur once every 1,000 to 10,000 nucleotide additions. To fix these, the cell leverages enzymes and pathways involved in lagging strand Okazaki fragment processing. After RER, the ribonucleotide insertion rate decreases by, again, around 99-99.9% or to one insertion every 100,000-1 million nucleotides. Most of those that get through are fixed by a version of the RER pathway that uses all the same enzymes but occurs outside the replisome.