.:Research
Viral Vectors for Gene Therapy
DNA Repair
DNA damage threatens to compromise the integrity of a cell’s genome. A variety of repair strategies have evolved and the specific strategy mobilized depends on the type of damage experienced. Whenever possible, cells use the undamaged sister chromatid as a template to recover the lost information.
Damage to DNA alters the host cell chromatin and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules are recruited to the site of damage, inducing other molecules to bind and form a complex that eventually effects the actual repair.
Thymidine dimers and methylation
Certain DNA repair mechanisms do not require a template, since the types of damage they fix can only occur in one of the four bases. The formation of thymine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent thymidine bases. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase.
Single strand damage
When only one of the two strands of a double helix experiences damage, the other strand can be used as a template for correction. A number of repair mechanisms are designed to remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to the other (undamaged) strand.
Base excision repair repairs damage due to a single nucleotide caused by oxidation, alkylation, hydrolysis, or deamination. Nucleotide excision repair repairs damage affecting longer strands of 2-30 bases. This process recognizes bulky, helix-distorting changes such as thymine dimers as well as single-strand breaks (repaired with enzymes such UvrABC endonuclease). A specialized form of NER known as Transcription-Coupled Repair deploys high-priority NER repair enzymes to genes that are being actively transcribed. Mismatch repair corrects errors of DNA replication and recombination that result in mispaired nucleotides following DNA replication.
Double strand breaks
A type of DNA damage particularly hazardous type to dividing cells is a break to both strands in the double helix. Double-strand breaks (DSBs) in DNA can result from exposure of cells to agents such as ionizing radiation (IR) and drugs and are also generated from endogenous sources such as meiotic recombination. Mammalian cells possess two main pathways for DNA double-strand break repair (DSBR): homologous recombination and non-homologous end-joining (NHEJ).
A key component of the NHEJ machinery is the DNA-dependent protein kinase (DNA-PK) that consists of a heterodimeric DNA binding sub-unit (Ku) and a catalytic subunit (DNA-PKcs). This complex is thought to bind DSBs and facilitate the recruitment and activation of other NHEJ components, and enables the repair process. Another conserved multi-protein complex consisting of Mre11, Rad50 and Nbs1/nibrin is important for DSBR, as well as meiotic recombination and telomere maintenance. This complex is thought to be a central player in the human DNA damage response and may be responsible for linking DNA damage detection to cell-cycle checkpoints and DNA repair. Upon DNA damage the Mre11 complex forms nuclear foci at sites of damage, and it has been suggested to act as a sensor of the double-strand breaks. The NHEJ pathway acts to maintain genetic stability and hence defects in cellular responses to DSBs predispose to malignancy. This can be seen in human diseases such as Nijmegen breakage syndrome and ataxia-telangiectasia-like disorder (ATLD), where patients have mutations in the NBS1 and Mre11 genes respectively.
Protein phosphorylation is a principal method of signaling used by the cellular DNA damage response. The three related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM), ATM-Rad3-related (ATR), and DNA-PKcs all play important roles in the signal transduction pathways and repair process. Recent studies have begun to elucidate the cellular requirements for activation of ATM and ATR upon DNA damage. It has been proposed that ATM is activated by intermolecular auto-phosphorylation on Ser1981, which results in dimer dissociation and enables phosphorylation of substrate proteins. In the case of ATR it has been suggested that RPA bound to single-stranded DNA is required for recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. DNA-PKcs is stimulated by double-strand ends and also becomes auto-phosphorylated. Although it may not play a central role in signaling to the cell cycle machinery, it may play an important role in signaling for cell death and has been suggested to be involved in mounting an innate response to bacterial and viral infection.
Many important DNA repair and checkpoint proteins have been identified as substrates for ATM and ATR kinase activity. These substrates include the downstream checkpoint kinases Chk1 and Chk2, and other damage response proteins such as 53BP1, H2AX, BRCA1, p53 and RPA32. DNA repair cascade pic.
Translesion synthesis
Translesion synthesis is an error-prone method of repairing a DNA lesion that has escaped repair by other mechanisms. The DNA replication machinery cannot continue replicating past a site of DNA damage, so the advancing replication fork will stall at the damaged base. The translesion synthesis pathway is mediated by specific DNA polymerases that insert extra bases at the site of damage and thus allow replication to bypass the damaged base to continue with chomosome duplication.
