III. Detailed Examination of the Mechanism of Replication A. Initiation B. Priming C. Elongation D. Proofreading and Termination

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1 Outline for Replication I. General Features of Replication A. Semi-Conservative B. Starts at Origin C. Bidirectional D. Semi-Discontinuous II. Proteins and Enzymes of Replication III. Detailed Examination of the Mechanism of Replication A. Initiation B. Priming C. Elongation D. Proofreading and Termination

2 DNA replication with two forks

3 DNA replication Fork The polarity of DNA synthesis creates an asymmetry between the leading strand and the lagging strand at the replication fork

4 Protein complexes of the replication fork: DNA primase DNA Helicase ssdna binding protein DNA Ligase DNA Topoisomerase DNA polymerase RNaseH Sliding Clamp Clamp Loader

5 Replication caught in Action Topoisomerase

6 II. Identifying Proteins and Enzymes involved in Replication A. Combine Genetics and Biochemistry 1. Genetic Approach: Obtain Mutants that are defective in Replication a. Such mutations are Lethal! b. Conditional lethal c. Temperature sensitive (ts) lethal 2. Method: a. Mutagenize cells b. Plate the cells on agar plates and grow at 30 o C c. Replica plate and grow at 37 o C

7 2 classes of DNA replication mutants identified by Francis Jacob et al 1. Quick-stop mutants: major class Stop replication immediately upon increase in temperature. Defect in elongation enzymes, enzyme that makes precursors 2. Slow-stop mutants: small class Complete current round of replication but cannot start next one > Replication initiation defect

$temperature and complement with wt components to identify dna defect ts mutant WT (fraction X) DNA DNA synthesis Purify DnaX component from fraction X$

8 In vitro complementation: A technique used to identify the mutants in DNA Replication (dna mutants) Make extract from dna mutant grown at restrictive temperature and complement with wt components to identify dna defect ts mutant WT (fraction X) DNA DNA synthesis Purify DnaX component from fraction X 37C

9 Priming of DNA replication Examples: Replication Rolling circle Replication Protein A nicks X174 viral strand at ori

10 DNA primase Primase is a special RNA polymerase that makes RNA primer on a ssdna template Unlike DNA Polymerase, primase can start synthesis de novo Makes an RNA primer of 4-12 nucleotides Different from other RNA polymerase: no sequence specificity How is it regulated? Activated only when bound by DNA replication proteins like helicases PRIMOSOME: complex formed between primase and helicase DnaG is primase in E. coli; it associates with RF proteins at oric Primase is not rifampicin sensitive while RNA pol is sensitive EXCEPTION: M13 phage uses bacterial RNA pol for primer synthesis Yeast: PRI1 and PRI2 Humans: primase (PRIM1, 2) Primase also acts as a halting mechanism to prevent the leading strand from outpacing the lagging strand by halting the progression of the replication fork. LEADING STRAND synthesis needs only 1 RNA primer LAGGING STRAND requires multiple ( ) RNA primers

11 DNA Primer synthesis On Lagging strand

12 DNA Replication at the leading strand Lagging strand

13 DNA Helicase (essential for cell survival) separates or unwinds DNA strands in a double-helix using ATP hydrolysis (1 ATP/bp unwound); thus an ATPase Thus creates ssdna templates for replication Functions as a hexamer Forms a ring and translocates along ssdna Unidirectional (5 to 3 ) Acts processively: helicase remains bound to substrate for long time DnaB is helicase in E. coli (12 helicases in all): identified as quick stop mutant Yeast and humans: Mcm complex (2-7)

14 Structure of a DNA Helicase DNA replication fork and helicase to scale. (C) Detailed structure of the bacteriophage T7 replicative helicase,

15 DNA Helicase Assay

16 SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

17 SSB (essential gene) Binds and maintains templates in single-stranded state Binds ssdna as tetramer Sequence-independent binding Co-operative binding: binding of 1 SSB to DNA promotes binding of another SSB by inter-ssb interaction; ensures rapid coating of ssdna SSB binding makes ssdna into an extended conformation which inhibits formation of hydrogen bonds Types: SSB in E. coli Yeast and humans: RPA (Replication Protein A); heterotrimeric protein

18 Structure of human SSB Proteins bound to DNA DNA

19 Topoisomerases DNA unwinding by helicase creates positive supercoils ahead of Replication forks The supercoiling ahead of the fork needs to be relieved or tension would build up (like coiling as spring) and block fork progression.

22 Supercoiling is relieved by the action of Topoisomerases. Type I topoisomerases: no ATP required Make nicks in one DNA strands Can relieve supercoiling Type II topoisomersases: ATP-dependent reaction Make nicks in both DNA strands (double strand break) Can relieve supercoiling and untangle linked DNA helices Both types of enzyme form covalent intermediates with the DNA E. coli: Gyrase (TopoII-like enzyme), TopoI Eukaryotes: both I and II function in replication

23 DNA topoisomerase I

24 DNA topoisomerase II

primers; this leaves gaps that are filled in by DNA polymerase

25 RNAseH: A special DNA repair enzyme that recognizes an RNA strand in an RNA/DNA hybrid and degrades it; thus removes RNA primers; this leaves gaps that are filled in by DNA polymerase Nicks in phosphate backbone are sealed by DNA ligase. RNAseH from E.coli

26 DNA Ligase: Uses ATP energy to make phosphodiester bond 2-step reaction: 1. ligase-amp complex forms 2. AMP complex attaches to 5 phosphate at the nick then a phosphodiester bond is formed with the 3'-OH terminus of the nick, thus releasing AMP and enzyme.

27 Mammalian replication Fork (eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

BIOCHEMISTRY REVIEW. Overview of Biomolecules. Chapter 11 DNA Replication

BIOCHEMISTRY REVIEW. Overview of Biomolecules. Chapter 11 DNA Replication BIOCHEMISTRY REVIEW Overview of Biomolecules Chapter 11 DNA Replication 2 3 4 5 6 7 8 9 Are You Getting It?? Which characteristics will be part of semi-conservative replication? (multiple answers) a) The