23. Errors II: Ribonucleotides (933) DONE

Surprisingly, the most common DNA polymerase error is not a mismatch at all. It's the insertion of a ribonucleotide, an RNA monomer, into a growing chain instead of a DNA monomer.

When that occurs, the bases pair correctly. But the small chemical hydroxyl (-OH) group on the ribose sugar alters the structure of the DNA backbone and makes it more prone to cleavage, increasing the risk of strand breaks if not removed.

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')

How often does this occur? Recall that the deoxyribonucleotide mismatch error rate after polymerase proofreading but before MMR was one error every 10 million to 100 million nucleotides.

In stark contrast, the ribonucleotide insertion rate is roughly one every 1,000 to 5,000 nucleotides in human cells! Relatively speaking, the cell is constantly mis-incorporating ribonucleotides,

There are two reasons for this. First, DNA polymerase's ability to discriminate the two isn't perfect; they differ only by one small chemical attachment (see figure).

Second, ribonucleotides are present in the nucleoplasm at 20-100 times higher concentration than deoxyribonucleotides. They're the raw materials of mRNAs, which the cell manufactures constantly to build proteins. Because they're so ubiquitous, they're easily grabbed and inserted.

Given how frequently ribonucleotides are inserted, the cell must remove them very effectively. It does so through a pathway called ribonucleotide excision repair (RER).

Ribonucleotide Excision Repair (RER)

Like MMR, RER is a modified cut-and-patch pathway, which consist of five similar steps.

First, a lesion is detected and flagged by a detector protein. A nick is then made by an endonuclease on the strand containing the error. The nick allows that strand, including the error, to be chewed back by an exonuclease. DNA polymerase then replaces the DNA near the now-excised error using the sequence from the other strand. The two abutting single-strands are then re-connected by a DNA ligase to restore strand continuity.

Because it is likewise focused on ribonucleotides, RER uses many of the same proteins that remove the RNA portions of Okazaki fragment primers in lagging strand synthesis. And like Okazaki fragment processing, the required proteins are replisome-linked. RER occurs right after DNA synthesis just behind the replisome.

RER starts with a familiar detector protein, RNase H2. Recall that RNase H2 was the enzyme that made endonucleolytic nicks to remove most of the first 10 or so ribonucleotides at the 5' end of Okazaki fragment primers.

In RER, the enzyme has two jobs. It recognizes ribonucleotides embedded in duplex DNA. It then uses a second enzymatic activity--an endonuclease activity--to make a nick just 5' of the ribonucleotide. Thus, in RER, RNase H2 performs both the first step of cut-and-patch repair (identifying the error) and the second step (making a single strand nick).

Once the nick is made, the PCNA sliding clamp recruits DNA polymerase delta. Here RER diverges from MMR.

In MMR, the next step was to chew up, or excise, the strand with the wrong DNA monomer. But RER is more like Okazaki fragment processing. The DNA polymerase delta just starts synthesizing at the nick using the good strand as the template.

As it synthesizes, it displaces the strand containing the ribonucleotide and several of the deoxyribonucleotides that follow, creating a flap.

The flap will end up being 1-10 nucleotides long. It will be cleaved off by FEN1 (Flap Endonuclease I), the enzyme from Okazaki fragment processing.

Once FEN1 cleaves off the flap, only one more job remains: connecting, or ligating, the two single strands. That falls to another familiar enzyme: 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.

RER, like MMR, is tightly coupled to DNA replication. As with MMR, all the enzymes involved in RER--RNase H2, DNA polymerase delta, FEN1, and DNA ligase I--tether to the back face of the PCNA sliding clamp .

Because ribonucleotide mis-incorporations are so common, RER is an obligatory quality control step in a DNA manufacturing process. RER removes the vast majority (about 99-99.9%) of incorporated ribonucleotides during S phase. The remaining ribonucleotides are handled later by other repair mechanisms.

A Complete Error Correction Solution

Together, proofreading, mismatch repair, and ribonucleotide excision repair form a layered quality-control system that reduces billions of potential copying mistakes to only a handful per genome.

The first layer is the DNA polymerase's own proofreading ability. More often than not, the enzyme senses an errant nucleotide and uses its 3' to 5' exonuclease activity to eliminate it.

Deoxyribonucleotide mis-incorporation errors and indels that get past proofreading will be sensed by MutS-alpha and repaired within the replisome via MMR, an example of a cut-and-patch pathway.

Finally, the most common kinds of DNA polymerase error are ribonucleotide mis-incorporations. To fix these, the cell uses the RER pathway, which borrows enzymes from lagging strand Okazaki fragment processing.

DNA polymerase errors are only one source of genomic insult. The others are lesions — chemical injuries to DNA that arise independently of replication and can be far more disruptive.

We turn next to these lesions and the remarkable repair systems that keep them in check so they don't interfere with genome replication.