9.7: DNA Repair Mechanisms

Many enzymes and proteins are involved in DNA repair, including eleven out of fourteen DNA polymerases in humans! Three DNA polymerases are for replication). Some of these function in normal replication, mitosis and meiosis, but were co-opted for DNA repair activities. These molecular co-optations are so vital to normal cell function that some repair activities and molecular players are highly conserved in evolution. Among the DNA repair pathways that have been identified, we will look at base excision repair, nucleotide excision repair, transcription-coupled repair, nonhomologous end-joining and homologous recombination. Of these, the last is perhaps the most complex.

9.7.1 Base Excision Repair

On detection of an error (e.g., oxidization, an open-ring, deamination, or bases containing saturated C=C bonds), DNA glycosylases catalyze hydrolysis of the damaged base from its deoxyribose. For more on these enzymes, see DNA Glycosylases. Figure 9.20 shows base excision repair.

Figure 9.20: In Base Excision Repair, (1) a glycosylase removes the sugar from the deaminated nucleotide; (2) an endonuclease nicks the strand at that nucleotide; (3) a DNA polymerase adds back a correct nucleotide and a DNA ligase seals the DNA strand to complete the repair.

9.7.2 Nucleotide Excision Repair

The discovery of nucleotide excision repair earned Aziz Sancar a share of the 2015 Nobel Prize in Chemistry. Figure 9.21 illustrates nucleotide excision repair for a pyrimidine dimer.

Figure 9.21: In nucleotide excision repair, (1) a fragment containing the damaged DNA, e.g., a pyrimidine (C-T) dimer is excised; (2) a polymerase fills in the missing nucleotides in the gap, and (3) a DNA ligase seals the remaining nick, resulting repaired DNA strands.

In this example, an Excision Nuclease recognizes a pyrimidine dimer, where it hydrolyzes phosphodiester linkages between nucleotides several bases away from either side of the dimer. A DNA helicase then unwinds and separates the DNA fragment containing the dimerized bases from the damaged DNA strand. Finally, DNA polymerase acts 5’ – 3’ to fill in the gap, and DNA ligase seals the remaining nick to complete the repair.

9.7.3 Mismatch Repair

DNA mismatch repair occurs when the proofreading DNA polymerase misses an incorrect base insertion into a new DNA strand. This repair mechanism relies on the fact that doublestranded DNA shows a specific pattern of methylation. The discovery of the mismatch repair mechanism earned Paul Modrich a share of the 2015 Nobel Prize in Chemistry. These methylation patterns are related to epigenetic patterns of gene activity and chromosome structure, which are expected to be inherited by daughter cells. When DNA replicates, the methyl groups on the template DNA strands remain, but the newly synthesized DNA is unmethylated. In fact, it takes some time for methylation enzymes to locate and to methylate the appropriate nucleotides in the new DNA. In the intervening time, several proteins and enzymes can detect inappropriate base pairing (the mismatches) and initiate mismatch repair.

The basic DNA mismatch repair process is illustrated in Figure 9.22.

Figure 9.22: Repair of a base mismatch in DNA begins when two Mut proteins (MutS and Mut) detect the mismatch (1); a third Mut protein (MutH) catalyzes cleavage at a methylated GATC site some distance from the mismatch (2), removing the mismatch (3); DNA polymerase III replaces the missing bases around the original mismatch (4); DNA ligase completes the repair (5).

Presumably, after mismatch repair is complete, the unmethylated new DNA is appropriately remethylated.

9.7.4 Transcription-Coupled Repair (in Eukaryotes)

If an RNA polymerase reading a template DNA encounters a nicked template or one with an unusual base substitution, it might stall transcription and seemingly “not know what to do next.” A normal transcript would not be made, and the cell might not survive. That’s no big deal in a tissue comprised of thousands if not millions of cells, right? But nevertheless, Transcription Coupled Repair exists!

In this repair pathway, if RNA polymerase encounters a DNA lesion (i.e., damaged DNA) while transcribing a template strand, it will indeed stall. This allows time for coupling proteins to reach the stalled polymerase and to enable repair machinery (e.g., by base or nucleotide excision) to make the repair. Once the repair is complete, the RNA polymerase “backs up” along the template strand (with the help of other factors) and resumes transcription of the corrected template.

9.7.5 Nonhomologous End-Joining

DNA replication errors can cause double-stranded breaks, as can environmental factors (ionizing radiation, oxidation). Repair by nonhomologous end-joining deletes damaged and adjacent DNA and rejoins the “cut” ends. When a double-stranded break is first recognized, nucleotides are hydrolyzed from the ends of both strands at the break-site, leaving “blunt ends.” Ku among other proteins then bring DNA strands together and further hydrolyze single DNA strands, creating overlapping staggered (complementary or sticky) ends. The overlapping ends of these DNA strands form H-bonds. Finally, DNA ligase seals the H-bonded overlapping ends of DNA strands, leaving a repair with deleted bases. Repair by nonhomologous end-joining is illustrated in Figure 9.23.

Figure 9.23: During Non-homologous End-Joining, endonucleases find and trim back DNA at the break (1); specific proteins bind to the trimmed ends of the DNAs (2); catalysis of single-strand trimming forms complementary (sticky) ends that overlap the break site (3). After H-bonds form between the staggered sticky ends (4), a DNA ligase seals the ends of the overlapping DNA strands (4) completing an effective repair, albeit with a deletion.

In older people, there is evidence of more than two thousand “footprints” of this kind of repair per cell. How is this possible? This quick-fix repair often works with no ill effects because most of the eukaryotic genome does not encode genes or even regulatory DNA (the damage of which would be more serious).

9.7.6 Homologous Recombination

Homologous recombination is a complex but normal, frequent part of meiosis in eukaryotes. Recall that homologous recombination occurs in synapsis in the first cell division of meiosis (meiosis I). During synapsis, alignment of homologous chromosomes may lead to DNA breakage, an exchange of alleles, and ligation to reseal newly recombinant DNA molecules. Novel recombinations of variant alleles in the chromosomes of sperm and eggs ensure genetic diversity in species. The key point is that DNA breakage is required to exchange alleles between homologous chromosomes. Consult the genetics chapter in an introductory biology textbook or the recombination chapter in a genetics text to be reminded of these events.

Cells use the same machinery to reseal DNA breaks during normal recombination and to repair DNA damaged by single- or double-stranded breakage. A single DNA strand that is nicked during replication can be repaired by recombination with the strands of homologous DNA that are being replicated on the other strand. A double-stranded break can be repaired using the same recombination machinery that operates on sister chromatids in meiosis. In both cases, the process accurately repairs damaged DNA without any deletions. These mechanisms are conserved in the cells of all species, further demonstrating an evolutionary imperative of accurate repair to the survival of species, no less important than the imperative to maintain the genetic diversity of species.

9.7.6.a Repair of a Single-Stranded Break

When a replisome reaches a break in one of the two strands of replicating DNA, the damage must be repaired, and the replication fork (RF) must be reestablished. Figure 9.24 illustrates the example of homologous recombination to repair a single strand break at a replication fork.

Figure 9.24: Single-stranded break repair by homologous recombination: An RF reaches a break in the left template strand (1), template DNA behind the break is trimmed back (2), while the 3’ end of the new DNA strand invades the new, (complementary) DNA strand replicating on the homologous parental template DNA and continues replicating (3). The missing DNA at the original break is re-synthesized (4). homologous recombination accurately repairs the break (5).

Such a break may have occurred prior to replication itself. Repair begins when the RF reaches the lesion. In the first step, a 5′-3’ exonuclease trims template DNA back along its newly synthesized complement. Next, RecA protein monomers (each with multiple DNA-binding sites) bind to the single-stranded DNA to form a nucleoprotein filament. With the help of additional proteins, the 3’ end of the filament scans the other replicating strand for homologous sequences. When such sequences are found, the RecA-DNA filament binds to the homologous sequences, and the filament of new DNA “invades” the homologous (i.e., opposite) double-stranded DNA, separating its template from its newly replicated DNA. After this strand invasion, the replication of the leading strand continues from the 3’ end of the invading strand. A new RF is established as the leading-strand template is broken and re-ligated to the original break. New lagging-strand replication then resumes at the newly rebuilt RF. The result is an accurate repair of the original damage, with no deletions or insertions of DNA.

RecA is a bacterial protein, an example of another one of those evolutionarily conserved proteins. Its homolog in the archaea is called RadA. In eukaryotes, the homolog is called Rad51, and it initiates synapsis during meiosis. Thus, it seems that a role for RecA and its conserved homologs in DNA repair predated its use in synapsis and crossing over in eukaryotes! For more about the functions of RecA protein and its homologs, see The Functions of RecA.

9.7.6.b Repair of a Double-Stranded Break

Homologous recombination can also repair a double-stranded DNA break with the aid of several enzymes and other proteins. Alternate repair pathways are summarized in Figure 9.25 (below). Here is a list of proteins involved in these homologous-recombination pathways:

• MRX, MRN: proteins that bind at double-stranded break and recruit other factors
• Sae2: an endonuclease active when phosphorylated to hydrolyze internal phosphodiester linkages
• Sgs1: a helicase that unwinds DNA that is under repair at a damaged RF
• Exo1, Dna2: single-strand exonucleases that hydrolyze terminal phosphodiester linkages
• RPA, Rad51, DMC1: proteins that bind to overhanging DNA to form a nucleoprotein filament and then initiate strand invasion at similar sequences

The activities of other enzymes in Figure 9.25 (below) are also identified. But not shown in the illustration are products of the BRCA1 and BRCA2 genes which binding to Rad51 (the human RecA homolog). Expressed mainly in breast tissue, their wild-type (normal) protein products participate in homologous recombination repair of double-stranded DNA breaks.

When mutated and dysfunctional, BRCA1 and BRCA2 genes increase the likelihood of a woman getting breast cancer due to uncorrected DNA damage in breast cells. It doesn’t help matters that the normal BRCA1 protein also plays a role in mismatch repair—and that the mutated protein can’t!

To end this chapter, here is a bit of weird science! Consider the tardigrade, a tiny critter that can survive the vacuum in space, along with assorted forms of radiation, resisting DNA damage, and failing that, rapidly repairing DNA damage (DNA Repair of the Tardigrade Genome).

Figure 9.25: In repairing a double-stranded DNA break, MRX or MRN proteins detect the break, after which several proteins (including exonuclease) enable trimming the strands at the break to make 'sticky ends' ((1). Other proteins, including Rad51, enable strand invasion while DNA polymerase replicates the invading strand (2). Alternate mechanisms called second end capture (3a) or strand displacement (3b) lead either to branch migration to resolve interlocking replicating DNA strands (4a), followed by homologous recombination to complete the repair (5), or to DNA synthesis and ligation to complete the repair without recombination (4b).