Lecture 21 - DNA Replication

Lecture 21: DNA Replication

  • Chapter 25

Lecture Overview

  • DNA Replication

  • DNA Repair

  • DNA Recombination

DNA Replication

  • Fundamental role in stable storage of genetic information.

  • DNA is a dynamic structure: it not only stores genetics but can be copied and modified.

  • New copies of DNA are synthesized with high fidelity before cell division.

  • Errors during synthesis must be repaired; segments of DNA can also rearrange through recombination.

  • Watson and Crick proposed a copying mechanism for DNA in their 1953 paper.

The Meselson-Stahl Experiment

  • Demonstrated that DNA replication is semiconservative.

  • Procedure:

    • Cells grown on medium containing only heavy nitrogen (15N) until DNA became fully labeled, producing a single band when centrifuged.

    • Cells switched to light nitrogen (14N), allowed to divide once, resulting in a medium band of dsDNA containing both heavy and light nitrogen.

    • Continued division resulted in a band showing only light nitrogen.

DNA Synthesis

  • Occurs in a 5’ to 3’ direction by adding new nucleotides to the 3’ end (3’-OH).

  • Leading Strand: synthesized continuously as replication fork advances.

  • Lagging Strand: synthesized in short pieces (Okazaki fragments) that are later ligated together.

  • Replication is bidirectional in circular DNA, with the presence of leading and lagging strands.

DNA Polymerase

  • Type of enzyme known as a transferase with three domains:

    • Palm: catalytic site with pre/postinsertion site.

    • Fingers: brings incoming bases into the active site.

    • Thumb: holds the polymerase to the DNA.

  • Polymerases have varying processivity: the measure of DNA length they can copy before dissociating.

DNA Elongation Chemistry

  • Parental DNA strand serves as a template in synthesis.

  • Deoxyribonucleotide triphosphates (dNTPs) are substrates for strand synthesis, incorporating nucleotides.

  • The nucleophilic 3’-OH group attacks the alpha-phosphate of the incoming trinucleotide, aided by Mg2+ ions.

  • Pyrophosphate (composed of beta and gamma phosphates) acts as a good leaving group and is further cleaved, driving the reaction forward.

Base Pairing Geometry

  • Ensures high fidelity during DNA replication.

  • DNA polymerase does not detect bases but uses the geometry of base pairs for accurate incorporation.

  • The active site of DNA polymerase excludes base pairs with incorrect geometries.

  • Errors occur at a rate of 1 in 10^4 to 10^5; however, repair mechanisms can correct mistakes.

  • Error rate in E. coli is around 1 in 10^9 to 10^10 base pairs.

Proofreading

  • Most DNA polymerases possess proofreading activity.

  • 3’ to 5’ exonuclease activity allows DNA polymerase to remove mismatched base pairs.

  • Synthesis pauses until the incorrect nucleotide is excised.

E. coli DNA Polymerases

  • DNA Polymerase I: Abundant (~400/cell), slow at 600 nucleotides/min, primarily involved in cleanup and repair.

  • DNA Polymerase III: Principal enzyme for replication.

  • DNA Polymerases II, IV, and V: Involved in DNA repair processes.

DNA Polymerase III

  • Holoenzyme composed of 10 subunits.

  • Two core domains connected by tau protein of the clamp loader complex.

  • Core domains interact with a dimer of beta sliding clamps to increase processivity (>500,000 bp).

  • E. coli replication involves more than 20 proteins collectively known as the replisome, including helicases, topoisomerases, and ligases.

Initiation of Replication

  • oriC (245 bp): AT-rich, recognized and bound by DnaA (ATPase).

  • DnaA bends DNA; DnaB helicase (hexamer) unwinds strands after binding DnaC.

  • DNA polymerase III attaches to DnaB, while single-strand binding proteins (SSBs) stabilize unwound strands.

  • Topoisomerase alleviates torsional strain ahead of the replication fork.

Regulation of Initiation

  • Replication must occur once per cycle.

  • DnaA dissociates after its ATP is hydrolyzed, then reassociates after 20-40 minutes.

  • After replication, oriC is hemimethylated at GATC by Dam methylase, recruiting SeqA.

  • SeqA's dissociation allows for full methylation of oriC for a subsequent replication round.

Elongation

  • Primase synthesizes RNA primer in association with helicase.

  • Primer is transferred to clamp loader, which then adds the sliding clamp and facilitates DNA polymerase III action.

  • Primer removal, gap filling by DNA polymerase I, and sealing of the nick by DNA ligase complete the process.

The Replisome

  • Continuous synthesis occurs on the leading strand.

  • DnaB helicase unwinds DNA, while the clamp-loading complex manages sliding clamps for Okazaki fragment synthesis on the lagging strand.

DNA Ligase

  • After removal of RNA primers, gaps are filled by DNA polymerase.

  • DNA ligase seals nicks by activating the 5’-P with AMP, enabling sealing through nucleophilic attack by a 3’-OH.

Termination

  • Replication forks converge at a region with 20-bp sequences (Ter), preventing fork movement.

  • Tus protein binds at Ter, halting the replication fork.

  • Eukaryotic linear DNA experiences shortening with each replication due to telomere dynamics.

Eukaryotic Replication

  • More complex than prokaryotic replication.

  • Yeast have ~400 origins in ranges of 30-300 kb, ensuring the entire genome is replicated per cell cycle.

  • Regulated by cyclins and CDKs, with synthesis speeds of ~50 nucleotides/sec.

  • Origin recognition complex initiates helicase loading.

  • Multiple polymerases involved in leading and lagging strand synthesis:

    • DNA pol a/primase: creates primers.

    • DNA pol d (delta): synthesizes lagging strand.

    • DNA pol e (epsilon): synthesizes leading strand.

DNA Mutations and Repair

  • Chemical and physical insults to DNA (alkylation, deamination, radiation, etc.) damage genomic DNA.

  • DNA is repaired mostly using the undamaged strand as a template, but some mutations escape repair.

  • Accumulation of mutations can lead to cancer and disease; potentially half a million mutations arise per day in each cell.

  • Over 130 human repair proteins are involved in maintaining genomic integrity.

The Ames Test

  • Evaluates the mutagenic potential of compounds.

  • Uses a strain of S. typhimurium that cannot synthesize histidine, combined with rat liver extract to mimic metabolism.

  • Successful mutagens can cause missense or frameshift mutations, enabling bacterial growth.

Methylation of DNA

  • In E. coli, the newly synthesized strand remains unmethylated shortly after synthesis.

  • Replication errors must be located on the unmethylated strand, which undergoes methyl-directed mismatch repair.

Mismatch Repair

  • Mismatches from replication errors are repaired by removal mechanisms.

  • MutS recognizes mismatches.

  • MutL identifies the methylated GATC.

  • MutH cleaves the unmethylated strand and coordinates unwinding and degradation processes.

  • Replace the excised sequence with the help of DNA pol III and ligase.

Direct Repair

  • Bases damaged by light or alkylation are repaired by directly reversing such modifications.

  • Photolyase utilizes light energy to repair pyrimidine dimers (absent in mammals).

  • Specific enzymes repair other damaged bases such as O6-methylguanine and 3-methylcytosine.

Base Excision Repair

  • Repairs incorrect or damaged bases via removal.

  • DNA glycosylases recognize specific lesions and break the glycosidic bond with the sugar, creating an AP site.

  • Glycosylases target bases like uracil and hypoxanthine.

Nucleotide Excision Repair

  • Removes larger distortions and bulky lesions (e.g., pyrimidine dimers).

  • Involves endonucleases (UvrA, UvrB, UvrC) in E. coli, cleaving the DNA backbone and removing 12-13 nucleotides.

  • DNA pol I and ligase are utilized to fill gaps.

Unrepaired ssDNA and dsDNA Breaks

  • Unrepaired lesions can stall replication forks.

  • Translesion synthesis uses specialized polymerases, part of the SOS response, allowing for mutagenic bypass of ssDNA lesions.

  • Homologous recombination repairs dsDNA breaks using complementary chromosomes as templates.

  • Nonhomologous end joining resolves dsDNA breaks by fusing strands, often causing mutations.

DNA Recombination

  • Segments of DNA can move within and between chromosomes.

  • Utilizes processes for DNA repair, homologous tension during meiosis, and allele swapping via crossing over.

  • Homologous Recombination: shares similar sequences.

  • Nonhomologous Recombination: occurs at incorrect sites.

  • Site-specific Recombination: takes place only at particular sequences.

  • DNA Transposition: short DNAs move from one chromosome to another.

Homologous Recombination

  • Key for DNA repair, chromosome segregation, and genetic diversity through allele shuffling.

  • Damaged template strands can cause replication forks to collapse, leading to double-stranded breaks.

  • The 5’-end is degraded, and the 3’-end is bound by recombinase for complementary invasion, forming a Holliday junction.

  • RecBCD processes broken DNA, unwinding and hydrolyzing until a chi sequence is reached to promote strand invasion.

RecA and Holliday Junction Resolution

  • RecA recombinase binds to the 3’ overhang, searching for homologous sequences.

  • RuvA binds at the junction where four arms converge.

  • RuvB hexamers use ATP to push DNA outward, while RuvC cleaves the Holliday junction to resolve recombination.

Meiosis and Genetic Diversity

  • Crossing over in meiosis maintains homologous chromosomes.

  • Double-stranded breaks at specific regions facilitate strand invasion and alignment.

Site-Specific Recombination

  • Specific short sequences govern this process; common in phage integrations and immunoglobulin recombination.

  • Utilizes recombinase enzymes that generate covalent bonds with DNA, allowing for inversions, deletions, and insertions.

Immunoglobulin Recombination

  • Human genome can produce many antibody types through recombination.

  • RAG proteins bind V(D)J segments, facilitating diverse antibody generation in B cells.

Transposons

  • Transposons can relocate within the genome, often carrying genes, including antibiotic resistance genes which can disrupt existing genes.

  • Retrotransposons use an RNA intermediate and reverse transcriptase to move (e.g., Alu, LINEs).

  • Notably, transposons comprise 44% of the human genome and 90% of maize genome.