DNA Replication and Synthesis

DNA Replication

• Primase rebinding to single-strand template DNA begins a new primer.
• Discontinuous DNA synthesis: the leading strand is extended continuously, while the lagging strand is synthesized in short fragments (Okazaki fragments) that are later joined by DNA ligase.

Proteins in Eukaryotic DNA Replication

• The structure of human DNA polymerase γ (pol γ) and T7 phage DNA polymerase holoenzymes show two unique domains (IP and AID) involved in DNA binding.

Fidelity of DNA Replication

• Telomerase adds simple repeat sequences to the 3’ end of telomeric DNA, allowing lagging-strand synthesis followed by ligation and RNA removal.
• The RNA carried by telomerase matches the 3’ DNA end, allowing its extension.
• The crystal structure of the telomerase catalytic subunit from the red flour beetle shows DNA and telomerase RNA modeled into the large central cleft.

DNA Repair

• DNA replication is not error-free, with a risk of error of 10-6 to 10-8.
• DNA repair systems correct errors in 99% of cases.
• Structures of pyrimidine dimer photoproducts, including thymine dimers, are formed when UV-irradiated DNA or DNA from a UV-irradiated organism is examined.
• DNA can be repaired directly or indirectly, by changing a damaged base to a normal one or replacing a DNA segment containing the damaged nucleotide.

Types of DNA Repair

• Direct repair: photolysis (use of light energy to repair pyrimidine dimers) and alkyltransferase (enzymes that are inactivated after one catalytic cycle)
• Indirect repair: single-strand damages (BER, NER, MMR) and double-strand breaks (NHEJ)

Alkylation Damage

• O6-methylguanine, a highly mutagenic product, can form a mispair with thymine, leading to a GC à AT transition.
• Repair of alkylation damage involves the enzyme O6-alkylguanine alkyltransferase, which transfers a methyl or ethyl group from an O6–methylguanine or O6-ethylguanine residue to a cysteine residue in the active site of the protein.

Oxidation Damage

• 8-oxoguanine, a strongly mutagenic alteration, forms readily with adenine and can lead to GC à AT or AT à GC transversions.
• MutT is a nucleotidase that cleaves 8-oxo-dGTP, the dNTP of 8-oxoguanine, to the corresponding nucleoside monophosphate, “sanitizing” cellular dNTP pools.
• MutM, mutT, and mutY gene products work together to counter the mutagenic effect of 8-oxoguanine.

Mismatch Repair

• Mutations in mismatch repair proteins were found in tumor cells from individuals with an inherited cancer predisposition called HNPCC (heritable nonpolyposis colon cancer).
• Germ-line mutations in the genes for five different mismatch repair proteins have been found to be associated with HNPCC.
• Tumor cells from those affected with HNPCC exhibit microsatellite instability, a large number of mutations in regions of the genome containing repeats of single-, double-, and triple-nucleotide sequences.
• DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination.

Nucleotide Excision Repair (NER)

• NER is a process for repairing DNA damage, including UV-induced damage.
• Replication fork regression is a likely response to a blocking DNA lesion in the leading strand template.