dna replication andy howard introductory biochemistry 8 may 2008

DNA replication

Andy HowardIntroductory Biochemistry

8 May 2008

08 May 2008DNA replication p.2 of 28

What we’ll discuss

Prokaryotic DNA replication Semiconservative

replication Unwinding of parent DNA Leading-strand replication Lagging-strand replication Okazaki fragments

Eukaryotic replication Rates Multiple

starting points Enzymes Other


iClicker quiz 1. Which of these DNA sequences

is palindromic? (a) TCGATG (b) TAGGAT (c ) GGCCCGGG (d) TACGCGTA (e) None of the above


iClicker quiz #2 Suppose someone introduced a drug that

interfered with deacylation of H1. What effect would this have on chromatin assembly? (a) It would prevent formation of the nucleosome

core particle (b) It would interfere with assembly of the

structures connecting one core particle to the next (c) Both (a) and (b) (d) It would have no effect on chromatin assembly (e) Like (c), except only in prokaryotes


Semi-conservative replication

A bit of a fanciful term Refers to the fact that, during DNA

replication, each daughter molecule contains one of the strands of the parent

Thus each daughter contains half (semi) of the original molecule

This mode of inheritance was predicted by the Watson/Crick model

Photo courtesy U. Costa Rica


Meselson & Stahl

1958: showed that DNA really is replicated this way

DNA grown with 15N has higher density 15N DNA allowed to replicate exactly once

has intermediate density


The E.coli chromosome One circular, double-stranded DNA

molecule of about 4.6*106 bp Replication begins in only one place, i.e. a

single origin of replication (OriC in E.Coli) Replication moves both directions until the

two replication efforts meet at the termination site

Protein machine that accomplishes replication is the replisome;one replisome in each direction

Replication forks move 1000 bp/sec; thus E.coli can be replicated in 38 min


By contrast: eukaryotes

Bigger chromosomes, more of them Not circular, usually Fruit-fly chromosomes:

Sex chromosome, 2 long autosomes,one tiny autosome

1.65 * 108 bp, 14000 genes Human

22 pairs of autosomes, sex chromosome 3.4*109 bp, ~22000 genes

Replication is bidirectional as in E.coli More than one origin so replication is

comparably fast even though rate is lower

Drosophila chromosomeTEM reconstruction


How replication works in prokaryotes

Takes place in the cytosol since there is no nucleus

Specific enzymes form the molecular machine to carry out the task

Has to involve separation of the strands Process divided into initiation,

elongation, and termination Enzymatic functions identified for each

segment


Prokaryotic DNA polymerases

Several varieties DNA polymerase III is the one responsible for

most of the work (but the 3rd discovered);it’s the biggest and most complex

DNA pol I involved in error correction and helps with replication of one of the strands

DNA pol II also does DNA repair Multi-subunit, complex entities


DNA Pol III Diagram from Kelman et al(1998) EMBO J. 17:2436


Components of DNA Pol III

Subunit Mr,kDa Gene Activity

130 polC/dnaE Polymerase

27 dnaQ/mutD 3’-5’ exonuclease

8.9 holE Stabilizes proofreading by ?

40 dnaN Sliding clamp

71 dnaX Dimer, ATPase

complex

15-47 Various Processivity


So how does it work? Add 1 nucleotide @ a time to 3’ end of growing chain Substrate is a dNTP Watson-Crick bp determines specificity Enzyme spends 75% of time tossing out wrong bases Forms phosphodiester linkage Pol III remains bound to the replication fork

Diagram from answers.com


Error correction in DNA pol III 3’-5’ proofreading recognizes incorrectly paired

bases and repairs most of them This is an exonuclease activity because it clips

off the last nucleotide in the chain 10-5 inherent error rate drops to 10-7 because the

exonuclease goofs 1% of the time Separate repair enzymes drop that down to 10-9


Processivity This term refers to the fact that many

nucleotides can be added to a growing chain following a single association event in which the polymerase (e.g. E.coli Pol III) associates with the template DNA.

We describe replication as highly processive if 50,000 bases can be replicated based on a single association of Pol III with our template.

subunits slide along, which is how this is done

complex is responsible for keeping the polymerase attached so that this is possible


Initiation

Begins in E.coli at a single origin called OriC DnaA binds to origin—region called DnaA box Replication fork forms after it binds Helicases & primases

set up for starting replication Complementary RNA tag attached at the

replication fork


Elongation

DNA polymerase operates in 5’-3’ directionon both strands

For one strand that’s straightforward replication moves in direction of unwinding of the DNA This is known as leading-strand synthesis

For the other strand it must work opposite to the unwinding therefore it’s more complicated This is lagging-strand synthesis


Termination Replication needs to know how

to stop Defined sequence ter is opposite

the origin on the chromosome Specific enzyme, Tus, involved in

recognizing termination signals Ter has sequences that play a

role in separating the daughter chromosomes

Tus-Ter complex;images courtesy Memorial Univ., Newfoundland


Leading-strand synthesis One base at a time is incorporated by subunit

of DNA polymerase, complementary to existing strand

At some point RNA primer is replaced with DNA

Image courtesy U.Pittsburgh


Lagging-strand synthesis

Movement of enzyme is opposite to unwinding Therefore it must work a few bases (~1000) at a

time and then back up The segments thus formed on the lagging

strand are known as Okazaki fragments DNA Pol I removes RNA primer DNA ligases link together Okazaki fragments


Primases These are DNA-dependent

RNA polymerase enzymes that initiate DNA synthesis, particularly on the lagging strand, where you need to do that at the beginning of each Okazaki fragment

DnaG helicasebinding domainPDB 2R6A347 kDa trimer ofheterotrimersBacillus stearothermophilus


Role of DNA Pol I

Part of the system for producing a continuous DNA strand on the lagging side

Contains both 5’3’ polymerase activity and 3’5’ proofreading exonuclease activity

Also has 5’3’ exonuclease activity: that’s used to remove the RNA primer


Rates and sequencing

Because there’s only one place where replication can begin, the process has to occur in discrete steps

The enzymes themselves are efficient, because they move with the unwinding of the double helix

Typical rates 1000 nucleotides/ sec So for E.coli it takes 38 min = 2280 sec to

replicate the entire chromosome(4.6*106 bp) / [(103 bp/sec)(2 directions)]=2300 sec


Where are things happening?

Both leading- and lagging-strand synthesis are catalyzed in both the clockwise and counterclockwise directions.

Each DNA Pol III molecule is catalyzing both leading- and lagging-strand synthesis.


Eukaryotic DNA polymerases

More complex, as you’d expect More than one initiation point Therefore even though the enzymes work less

rapidly, they can multiplex the process The result is that human DNA can be replicated

in roughly the same time scale as E.coli DNA

image courtesy Memorial Univ., Newfoundland


Eukaryotic DNA polymerases

Several complexes involved: Elongation and repair Elongation and repair Elongation and repair DNA repair Helps in replicating mitochondrial DNA chloroplast polymerase

Don’t fall into the trap: these Greek letters don’t necessarily mean the same thing that they mean in the context of bacterial replication!

Human DNA polymerase :From answers.com


Specific roles for and complex does leading-strand synthesis

and 3’-5’ exonuclease activity, which is impressively effective

and involved in lagging strand synthesis:

is DNA polymerase and RNA primase; extends segment to complete Okazaki fragment


Roles for DNA polymerase

Big,multi-subunit protein Similar to DNA pol I from E.coli It repairs and it fills gaps between

Okazaki fragments Largest piece is a polymerase

and does 3’5’ proofreading

Cryo EM image from Asturias et al (2006) Nature Struct Mol Biol 13:35;yeast

dna replication andy howard introductory biochemistry 8 may 2008

Documents

dna replicationwhat

dna sequences

dna repairmultisubunit

higher density15n dna

doublestranded dna molecule

replication efforts

complexdna pol

strandsdna pol