1. DNA Replication, Repair, and Recombination BIO 224 Intro to Molecular and Cell Biology

2. DNA Replication Proceeds in a semi-conservative manner Each strand acts as a template for synthesis of new complementary strand Meselson and Stahl’s experiments with 14 N and 15 N labeled DNA in E. coli provided evidence of replication method DNA polymerase is primary enzyme involved Other proteins involved, with different jobs

6. DNA Polymerases First identified in E. coli in 1956 Enzyme’s DNA copying ability gave chemical basis for replication method proposed by Watson and Crick DNA Pol I was identified first, not major enzyme in replication, more for repair Prokaryotes and eukaryotes have different DNA polymerases for replication and repair roles

8. DNA Polymerases Prokaryotes use DNA Pol I, II, and III Eukaryotes use DNA Pol α, δ, and ε in the nucleus, and γ in the mitochondria All synthesize DNA only in 5’ to 3’ direction Only add dNTPs to polymer to preformed primer H-bonded to the template Short segments of complementary RNA serve as primers to begin replication

9. Replication Fork Region of DNA where parent strands separate and synthesis of new daughter strands occurs One strand continuously copied (leading strand) and other strand (lagging strand) copied in short discontinuous pieces (Okazaki fragments) – Due to 5’ to 3’ only activity of DNA polymerase and antiparallel nature of DS DNA Leading strand synthesized in direction of movement of fork, lagging strand synthesized in opposite direction DNA ligase joins Okazaki fragments to complete lagging strand

13. DNA Synthesis Primase is an enzyme that synthesizes short RNA primers complementary to the lagging strand at the replication fork DNA polymerase uses exonuclease activity to remove RNA primers from Okazaki fragments in prokaryotes – Enzyme that hydrolyzes DNA molecules in either the 5 to 3 or 3 to 5 direction. RNase H is enzyme used by eukaryotes that degrades the RNA strand of RNA-DNA hybrids, and 5 to 3 exonucleases.

16. DNA Replication DNA polymerase interacts with accessory proteins to attach to and remain associated with primers and template DNA RFC (replication factor C) in eukaryotes and PCNA in prokaryotes (proliferating cell nuclear antigen) Helicases unwind parent DNA ahead of replication fork SS DNA binding proteins stabilize unwound template DNA and maintain single strands DNA rotates ahead of replication fork, topoisomerases catalyze reversible breakage and rejoining of DNA strands to relieve twisting

21. Replication Fidelity Accuracy of replication critical for cell reproduction Error frequency only 1 per 10 8 to 10 9 nucleotides DNA polymerase helps with selection of correct bases for pairing DNA polymerase has proofreading ability – Uses exonuclease activity to remove incorrectly incorporated base and allow for replacement with correct base

23. Origin and Initiation of Replication ORIs are sites made of specific sequences for initiator proteins to bind to and start replication process – Exact recognition mechanism still unknown in higher eukaryotes Prokaryotes have single ORIs; eukaryotes have many – Allow timely replication of large genomes Replication proceeds in both directions along chromosomes, from ORIs

26. Telomeres Repeats of simple sequence DNAs at chromosome ends Extreme 5’ ends of linear DNA can’t be copied by DNA polymerases Telomerase enzyme can synthesize telomeres without DNA template – Reverse transcriptase that copies DNA from RNA template Telomerase defects and deficiencies in telomere maintenance associated with human disease

28. Mutation Mutation can occur from incorporation of incorrect bases during DNA replication Various chemical changes can occur spontaneously, or from exposure to chemicals, radiation, viruses – Can block replication or transcription leading to high frequency of mutation potentially devastating to cells

33. DNA Repair Cells have mechanisms to repair damaged DNA by one of two general means: Direct reversal of the chemical reaction causing the change Removed of damaged bases and replacement with new DNA If repair fails, other mechanisms are available to cells for dealing with damage

34. Direct Damage Reversal Some damage repaired this way May be more efficient method Pyrimidine dimers resulting from UV light exposure repaired this way – Photoreactivation uses visible light energy to disrupt dimerization and return bases to original state Not found in humans Repair of alkylation of DNA by chemical agents directed by enzyme that removes the alkyl group and restores original base another type of direct reversal

37. Excision Repair More types of damage repaired this way General mechanism for wide variety of chemical modifications of DNA Damaged DNA recognized and removed as free bases or nucleotides, with gaps filled by new DNA strand copied from the other, undamaged, template strand

38. Excision Repair Base-excision repair removes and replaces single damaged bases Nucleotide-excision repair removes and replaces oligonucleotide containing damaged area Errors in this type of repair lead to disease – Transcription-coupled repair fixes damage in actively transcribed genes Mismatch repair finds bases added incorrectly during DNA replication (if missed by DNA polymerase proofreading)

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