DNA Replication and Repair

DNA Replication, Repair, and Recombination

• Germ-line cells propagate genetic information to the next generation.
• Gametes (e.g., eggs and sperm) have half the number of chromosomes as other body cells.
• Zygotes form after fertilization, containing a full set of chromosomes.
• Somatic cells form the organism's body and do not contribute their DNA to the next generation.

DNA Replication as a Template

• DNA acts as a template for its own replication.
• Nucleotides A pairs with T, and G pairs with C.
• Each strand of a DNA double helix can serve as a template for a complementary strand.
• Two exact copies of the original double helix are produced.

DNA Synthesis Chemistry

• Nucleotides enter as deoxyribonucleoside triphosphates.
• Addition of a deoxyribonucleotide to the 3' end of a polynucleotide chain is the fundamental DNA synthesis reaction.
• Base pairing between incoming and existing strands guides new strand formation.
• Ensuring complementary nucleotide sequence.

DNA Polymerase's Role

• DNA polymerase adds deoxyribonucleotides to the 3' end of a growing DNA strand.
• This occurs in the 5'-to-3' direction.
• Energy for polymerization comes from hydrolysis of high-energy phosphate bonds in incoming nucleotides.
• DNA polymerase guides the incoming nucleoside triphosphate to the template strand.
• DNA polymerase proofreads its work.
• If an incorrect nucleotide is added, DNA polymerase removes it and replaces it with the correct one.
• This process maintains high fidelity of DNA replication.

DNA Polymerases Contain Separate Sites

• DNA polymerases have separate sites for synthesis and proofreading.
• Polymerization activity is distinct from editing activity.
• Incorrect nucleotides are removed in the editing site.
• This process ensures nucleotide sequence accuracy.

Different Enzymes for Lagging Strand Synthesis

• RNA primers are synthesized by primase, using a DNA template.
• Primase synthesizes 5' to 3'.
• Primase can start a new polynucleotide chain without a base-paired 3' end.
• Eukaryotic RNA primers are made at intervals, typically 10 nucleotides long.
• These primers are extended by DNA polymerases.
• Primers are removed by nucleases.
• Gaps are filled in by repair DNA polymerase, with proofreading ability.
• Fragments are joined by DNA ligase.

DNA Helicase Enzymes and Strand Separation

• DNA helicases separate DNA strands.
• Their movement is powered by ATP hydrolysis.
• Most DNA helicases consist of a ring of 6 subunits.

Single-Stranded DNA-Binding Proteins (SSB)

• SSB proteins bind to single-stranded DNA.
• They prevent re-annealing of DNA strands.
• Cooperative binding helps straighten DNA, improving the process of DNA polymerization.
• Prevents hairpin helices from forming.

Eukaryotic Mismatch Repair

• In eukaryotes, MutS proteins bind to mismatched base pairs.
• They recruit MutL and the complex scans for a gap and a sliding clamp.
• MutL and another nuclease removes the mismatched portion of DNA.
• This is followed by DNA polymerase to fill in the gaps.
• Sealed by DNA Ligase.

DNA Topoisomerase I and II

• Type I DNA topoisomerases create a transient single covalent bond with DNA.
• This allows free rotation of the DNA around the covalent backbone.
• Type II DNA topoisomerases hydrolyze ATP to release and reset the enzyme.
• They are essential during rapidly dividing cells.

Replication Fork Initiation

• DNA replication starts at specific origin sites.
• Origin-recognition complex (ORC) binds to origins in eukaryotes.
• Initiator proteins destabilize DNA.
• Additional proteins participate in loading helicases.
• Primers are required and then synthesized.

Bacterial Replication Fork

• Bacterial chromosomes are circular.
• Two replication forks move in opposite directions.
• DNA is unwound and separated.
• New strands are synthesized.

Eukaryotic Replication Fork

• Unlike bacteria, eukaryotic replication forks can function independently.
• CMG (Cdc45-MCM-GINS) helicase moves unidirectionally along the leading strand template during replication.
• Eukaryotic DNA replication is more complicated.

Telomere Replication

• Telomeres are repetitive sequences at the ends of linear eukaryotic chromosomes.
• Telomerase synthesizes new telomere DNA by using its RNA molecule as a template.
• DNA polymerase then extends the strand.