Nucleotide Excision Repair, Photoreactivation & Recombination

Nucleotide Excision Repair

Nucleotide excision repair involves an enzyme complex that hydrolyzes phosphodiester bonds on one side of the distortion caused by the lesion. The damaged oligonucleotide is removed through the incisions, and the resulting gap is filled by DNA polymerase I in E. coli and by DNA polymerase ε in eukaryotes. The nick is then sealed by DNA ligase.

A protein complex formed by UvrA and UvrB scans DNA and binds to the site of the lesion. After the UvrA dimer dissociates, UvrB remains anchored to the strand. UvrC then binds to UvrB and makes the cuts in the phosphodiester backbone required. The excised fragment is removed by a helicase, and the gap is filled by DNA polymerase I and sealed by DNA ligase.

Various types of lesions are repaired without removing the damaged base or nucleotide. An example is the direct photoreactivation of cyclobutane pyrimidine dimers; this reaction is promoted by DNA photolyase. Pyrimidine dimers are produced by ultraviolet (UV) light, and photolyase uses absorbed light energy to reverse the reaction. Photolyase contains two cofactors (or chromophores) that act as light-absorbing agents; the mechanism involves electron transfer that can include transient free-radical intermediates.

Key Proteins and Steps

• UvrA–UvrB complex: scans and recognizes damage
• UvrA: dissociates after lesion recognition
• UvrB: remains bound and recruits UvrC
• UvrC: performs dual incisions
• Helicase: removes the excised oligonucleotide
• DNA polymerase: fills the gap
• DNA ligase: seals the nick

Photoreactivation and Photolyase

Photoreactivation is a direct repair mechanism for cyclobutane pyrimidine dimers mediated by DNA photolyase. Photolyase absorbs light via its chromophores and uses that energy to cleave the dimer, restoring the original bases without excision. The cofactors act as light-absorbing chromophores that enable the enzymatic reversal.

5) Site-Specific Recombination

Rearrangement of genetic information between DNA molecules comprises a number of processes.

Homologous and Site-Specific Recombination

Homologous recombination is genetic exchange between two DNA molecules involving a large region of nearly identical sequence. Site-specific recombination takes place only at particular, defined DNA sequences. DNA transposition is different from the previous two: it involves a short segment of DNA with the remarkable ability to relocate from one location to another on the chromosome or to a different chromosome.

If we focus on site-specific recombination, we can say that accurate DNA rearrangement occurs at defined sites, whereas homologous recombination can occur between any two sequences provided they are sufficiently similar.

Mechanism of Recombinases

All recombination systems described require a recombinase enzyme and unique DNA sequences at which the enzyme acts. A recombinase recognizes and attaches to each of the two recombination sites. At each site, it cuts a DNA strand at a specific point. The recombinase often becomes covalently bound to the DNA; this protein–DNA bond preserves the integrity of the phosphodiester backbone at the cut. Short DNA strands are exchanged to form a new connection (a Holliday-type exchange) and new phosphodiester bonds are formed.

To complete the reaction, the process must be repeated at a second point within each of the two recombination sites. In some systems, both strands at the cut site of recombination are cleaved simultaneously and the new strands are joined directly without an intermediate Holliday junction.