template base thumb nucleotide template base as much as 1,000-fold compared to a completely nonprocessive enzyme.
Increased processivity is facilitated by the ability of DNA polymerases tn slide along the DNA template. Once bound to a primer:template junction, UNA polymerase interacts tightly with much of the double-stranded portion of the DNA in a sequence nonspecific manner. These interactions include electrostatic interactions between the phosphate backbone and the "thumb" domain, and interactions between the minor groove of the DNA and the palm domain (described above). The sequence-independent nature of these interactions permits the easy movement of the DNA even after it binds to polymerase. Each time a nucleotide is added to the primer strand, the DNA partially releases from the polymerase [the hydrogen bonds with the minor groove are broken but the electrostatic interactions with tile thumb are maintained). The DNA then rapidly re-binds to the polymerase in 8 position that is shifted by one base pair using the same sequence nonspecific mechanism. Further increases in processivity are achieved through interactions between the DNA polymerase and a "sliding clamp" protein that completely encircles the DNA, as we shall discuss further below.
A system based only on base-pair geometry and the complementarity between the bases is incapable of reaching the extraordinarily high levels of accuracy that are observed for UNA synthesis in the cell (approximately 1 mistake in every 10)0 base pairs added). A major limit to DNA polymerase accuracy is the occasional (approximately once in 10s times) flickering of the bases into the "wrong" tautomeric form fimino or enol: see Figure 6-5), These alternate forms of the bases allow incorrect base pairs to be correctly positioned for catalysis. As we now describe, proofreading allows these mistakes to be corrected.
Proofreading of DNA synthesis is mediated by nucleases that remove incorrectly base-paired nucleotides. This type of nuclease was originally identified in the same polypeptide as the DNA polymerase and is now referred to as proofreading exonuclease. These exonucleases aie capable of degrading DNA starting from a 3F DNA end, that is from the growing end of the new DNA strand. (Nucleases that can only degrade from a DNA end are called exonucleases; nucleases that can cut in the middle of a DNA strand are called endonucleases.)
initially, the presence of a 3' exonuclease as pari of the same polypeptide as a DNA polymerase made little sense. Why would the DNA polymerase need to degrade the DNA it had just synthesized? The role for these exonucleases became clear when it was determined that they have a strong preference to degrade DNA containing incorrect base pairs. Thus, in the rare event that an incorrect nucleotide is added to the primer strand, the proofreading exonuclease removes this nucleotide from the 3' end of the primer strand. This "proofreading" of the newly added DNA gives the DNA polymerase a second chance to add the correct nucleotide.
The removal of mismatched nucleotides is facilitated by the reduced ability of DNA polymerase to add a nucleotide adjacent to an incorrectly base-paired primer. Mispaired DNA alters the geometry of the 3F-OH and the incoming nucleotide due to poor interac-
a stow or no DNA synthesis b removal at mismatched nuctaoUdes eitdfujcleasff dip**.
lions with the palm region. This altered geometry reduces the rate of nucleotide addition in much the same way that addition of an incorrectly paired dNTP reduces catalysis. Thus, when a mismatched nucleotide is added, it both decreases the rate of new nucleolide addition and increases the rain of proofreading exonucle-ase activity.
As with DNA synthesis, proofreading can occur without releasing the DNA from the polymerase (Figure 8-10). When a mismatched base pair is detected by the polymerase, the primer:template junction slides away from the DNA polymerase active site and into the exonucleasc site. (This is because the mismatched DNA has a reduced affinity of the palm region.) After the incorrect base pair is removed, the correctly paired primontemplate junction slides back into the DNA polymerase active site and DNA synthesis can continue.
In essence, proofreading exonucleasns work like a "delete key" on a keyboard, removing only the most recent errors. The addition of a proofreading exonuclease greatly increases the accuracy of DNA synthesis. On average, DNA polymerase inserts one incorrect nucleotide for every I0ri nucleotides added. Proofreading exonucleases decrease the appearance of an incorrect paired base to one in every H)7 nucleotides added. This error rate is still significantly short of the actual rate of mutation observed in a typical cell (approximately one mistake in every 1010 nucleotides added). This additional level of accuracy is provided by the post-replication mismatch repair process that is described in Chapter 9.
c resume UNA synthesis
FIGURE 8-10 Proofreading exonucleases removes bases irom the 3' end of mismatched DMA. (a) When an incorrect nucleotide is incorporated into the DNA by a polymerase, the rate o! DNA synthesis is reduced and the affinity of the 3'ervd oj the primer lor the DNA polymerase active site is diminished (b) When mismatched, the 3' end of the DMA has increased affinity for the proofreading exonudease active site Once bound at this active site, the mismatched nucleotide is removed, (c) Once the mismatched nucleotide ts removed, the affinity of the properly base-paired UNA for the DMA polymerase active site is restored and DNA synthesis continues, (Source: Adapted from Baker TA and Bell S.P 1996, Polymerases and the replisorne Machines within machines Cell 92 296, fig. ib. Copynght £■ 199B with permission from Bsewer.)
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