DNA Replication

DNA is the Genetic Material Therefore it must Replicate faithfully. Have the coding capacity to generate proteins and other products for all cellular functions. “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick

Replication Watson and Crick develop the double helix model of DNA

The Dawn of Molecular Biology April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." 3

Testing Models for DNA replication Matthew Meselson and Franklin Stahl (1958)

Models for DNA replication 1) Semiconservative model: Daughter DNA molecules contain one parental strand and one newly-replicated strand 2) Conservative model: Parent strands transfer information to an intermediate (?), then the intermediate gets copied. The parent helix is conserved, the daughter helix is completely new 3) Dispersive model: Parent helix is broken into fragments, dispersed, copied then assembled into two new helices. New and old DNA are completely dispersed

MODELS OF DNA REPLICATION (a) Hypothesis 1: (b) Hypothesis 2: (c) Hypothesis 3: Semi-conservative replication Conservative replication Dispersive replication Intermediate molecule

Meselson and Stahl Semi-conservative replication of DNA Isotopes of nitrogen (non-radioactive) were used in this experiment

Bases paired Strands antiparallel

TEMPLATING/REPLICATION REPLICATION OF INFORMATION

Key proposal of Watson and Crick: base pairs A : T and G : C are specific. Base pairing regulates replication.

Replication is a Process Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

Replication DNA molecule separates into 2 strands Each strand serves as a template for a new strand Complementary strand is made

Replication and Enzymes Enzymes = proteins helping to catalyze (start and speed up) a reaction Main enzyme in replication = DNA polymerase DNA polymerase Joins individual nucleotides to make DNA “proofreads” new DNA strands to check for error

DNA replication Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)

Replication can be Uni- or Bidirectional Origin 5’ 3’ UNIDIRECTIONAL REPLICATION Origin 5’ 3’ BIDIRECTIONAL REPLICATION

T7 DNA replication 1 Replicating bubble in DNA from bacteriophage T7 Two replication forks heading towards opposite ends of the DNA

Features of DNA Replication DNA replication is semiconservative Each strand of both replication forks is being copied. DNA replication is bidirectional Bidirectional replication involves two replication forks, which move in opposite directions

Arthur Kornberg (1957) Protein extracts from E. coli + Template DNA Is new DNA synthesized?? - dNTPs (substrates) all 4 at once - Mg2+ (cofactor) - ATP (energy source) - free 3’OH end (primer) In vitro assay for DNA synthesis Used the assay to purify a DNA polymerizing enzyme DNA polymerase I

-therefore DNA synthesis occurs only in the Kornberg also used the in vitro assay to characterize the DNA polymerizing activity - dNTPs are ONLY added to the 3’ end of newly replicating DNA 5’ 3’ Parental template strand New progeny strand 5’ 3’ 3’ 5’ 3’ 3’ 5’ 3’ 3’ -therefore DNA synthesis occurs only in the 5’ to 3’ direction

THIS LEADS TO A CONCEPTUAL PROBLEM Consider one replication fork: 5’ 3’ 5’ 3’ Primer Continuous replication Direction of unwinding Primer 5’ 3’ Discontinuous replication Primer 5’ 3’

Evidence for the Semi-Discontinuous replication model was provided by the Okazaki (1968)

Evidence for Semi-Discontinuous Replication (pulse-chase experiment) Bacteria are replicating Bacterial culture Add 3H Thymidine For a SHORT time (i.e. seconds) Flood with non-radioactive T Allow replication To continue Harvest the bacteria at different times after the chase smallest largest Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient Radioactivity will only be in the DNA that was made during the pulse

Results of pulse-chase experiment 5’ 3’ Direction of unwinding Primer smallest largest * Primer 5’ 3’ *

DNA replication is semi-discontinuous Continuous synthesis Discontinuous synthesis

Features of DNA Replication DNA replication is semiconservative Each strand of template DNA is being copied. DNA replication is bidirectional Bidirectional replication involves two replication forks, which move in opposite directions DNA replication is semidiscontinuous The leading strand copies continuously The lagging strand copies in segments (Okazaki fragments) which must be joined

The Enzymology of DNA Replication In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I DNA Polymerase I has THREE different enzymatic activities in a single polypeptide: a 5’ to 3’ DNA polymerizing activity a 3’ to 5’ exonuclease activity a 5’ to 3’ exonuclease activity 5

The 5’ to 3’ DNA polymerizing activity Subsequent hydrolysis of PPi drives the reaction forward Nucleotides are added at the 3'-end of the strand

Why the exonuclease activities? The 3'-5' exonuclease activity serves a proofreading function It removes incorrectly matched bases, so that the polymerase can try again. 7

Proof reading activity of the 3’ to 5’ exonuclease. DNAPI stalls if the incorrect ntd is added - it can’t add the next ntd in the chain Proof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.

DNA Replication is Accurate (In E. coli: 1 error/109 -1010 dNTPs added) How? 1) Base-pairing specificity at the active site - correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ 104 -105 dNTPs added 2) Proofreading activity by 3’-5’ exonuclease - removes mispaired dNTPs from 3’ end of DNA - increases the accuracy of replication 102 -103 fold 3) Mismatch repair system - corrects mismatches AFTER DNA replication

Why the exonuclease activities? The 5’-3' exonuclease activity is used to excise RNA primers in a recation called “nick translation” We will discuss this in the next lecture. 7

Is DNA Polymerase I the principal replication enzyme?? In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I activity (polA) - mutant was super sensitive to UV radiation - but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA Conclusion: DNAP I is NOT the principal replication enzyme in E. coli 9

Other clues…. - DNAP I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome instead of 40 minutes) - DNAP I is only moderately processive (processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template) Conclusion: There must be additional DNA polymerases. Biochemists purified them from the polA mutant 9

So if it’s not the chief replication enzyme then what does DNAP I do? - functions in multiple processes that require only short lengths of DNA synthesis - has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive) - its role in DNA replication is to remove primers and fill in the gaps left behind - for this it needs the nick-translation activity 9

The DNA Polymerase Family A total of 5 different DNAPs have been reported in E. coli DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999) DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999) DNAP V: functions in DNA repair (discovered in 1999) 9

The "real" replicative polymerase in E. coli DNA Polymerase III The "real" replicative polymerase in E. coli It’s fast: up to 1,000 dNTPs added/sec/enzyme It’s highly processive: >500,000 dNTPs added before dissociating It’s accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall. 9