dna replication andy howard introductory biochemistry 4 december 2008

DNA replication

Andy HowardIntroductory Biochemistry

4 December 2008

12/04/2008DNA replication p.2 of 43

DNA replication: accuracy!

The extraordinary fidelity of heritance in prokaryotes and eukaryotes derives from the net accuracy of DNA replication. We’ll outline the steps of replication and the proofreading that goes with it.


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

Repair & recombination Forms of repair Recombination Repair & Disease


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


3 models (fig. 28.3)


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


Meselson-Stahl experiment

Fig. 28.4;note that the

bottom 2 density

gradients are for mixtures

of generations


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 = 2280 s


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 & primasesset 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


iClicker question #33. A deleterious mutation in the gene coding for the subunit of DNA pol III would have what direct effect on replication in E.coli?

(a) it would increase error rates (b) it would decrease processivity (c ) it would prevent formation of Okazaki fragments

(d) none of the above


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 Polymerase 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 polymeraseand does 3’5’ proofreading

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


iClicker question 4

4. A deleterious mutation in eukaryotic DNA polymerase would directly affect

(a) trafficking of membrane-bound proteins from the ribosome to the plasma membrane

(b) assembly of the nucleosome core particle

(c) energy production in the cell (d) harvesting of light from the sun (e) there is no eukaryotic DNA polymerase


DNA repair DNA is the only macromolecule that gets repaired: it’s too important not to

A single base error can be fatal, even in prokaryotes

Natural rates of misincorporation are small but nonzero

Rate can go up upon exposure to ionizing radiation, some chemicals, some toxins


Direct repair

Enzymes scan DNA for particular lesions

Pyrimidine dimers are noted and repaired this way

Some can replace the base without breaking the phosphodiester backbone

Image courtesy U. München


Excision repair

Endonuclease recognizes lesion

Cleaves upstream &downstream —12-13 bases

Only cleaves damaged strand Removal may require helicase

DNA polymerase (I in prokaryotes) fills the gap

DNA ligase reseals the lesion

Diagram courtesy Beth Montelone, Kansas State U.


Other repairs Repairing hydrolytic deamination of A, C, G: DNA glycosylase flips base out and hydrolyzes glycosidic bond

Endonuclease sutures in one replacement base (sometimes part of same protein)

N

OHN NH2

cytosine

HN

OHN O

uracil

H2O

NH3


Recombination Recombination is any

exchange or transfer of DNA from one spot to another

Homologous recombination involves exchanges in closely-related sequences; can involve paired chromosomes

Transposons are elements that can be readily recombined nonhomologously


Holliday model Cf.fig.28.19

Involves nicking, then rotation

Can result in exchanging the ends of two homologous chromosomes


Recombination in E.coli

RecBCD endonuclease creates single-stranded DNA with free 3’

SS DNA invades double helix of neighboring DNA

RecA promotes strand exchange: Requires energy Formation of triple-

stranded intermediate Branch migrates down

chainMazin& Kowalczykowski,(1998) EMBO J. 17:1161


Recombination as repair Bad lesions are simply skipped

Intact strand from one daughter acts as template for repairing broken strand

Most recombination genes play roles in repair too

E.coli RecA in compressed helical form


Repair & Disease

Repair deficiencies render the organism susceptible to mutation-related maladies

BRCA1&BRCA2 are proteins that bind to RecA in humans and help repair DSBs

Therefore mutations in these proteins leave people prone to cancer

PDB 1t15:BRCA1 BRCT domains complexed to BACH1 helicase


Ataxia telangiectasia

Ataxia is lack of coordination Telangiectasia is spider-veins Condition involves both symptoms Caused by mutation in specific protein involved in cell-cycle control

Patients are abnormally prone to cancer, other DNA-repair related conditions

Ataxia telangiectasia involves failure to recognize DSBs ~1% population heterozygous 8-10% of breast cancer patients are heterozygotes for this condition; Swift (2001) JNCI 93:84

PDB 2csv

dna replication andy howard introductory biochemistry 4 december 2008

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