dna replication

DNA replication

醫技系楊孔嘉

References

1. Nature 2003, 421:p431 (replication & recombination)2. EMBO reports 2003, 4:p666 (replication) 3. BioEssays 2003, 25:p116 (MCM proteins)4. Gene 2003, 310:p1 (ORC cycle)5. FEMS Microbiol Rev 2003, 26:p533 (structure of OBPs)6. Cancer Lett 2003, 194:p139 (telomerase)7. J Struct Biol 2003, 140:p17 (initiation)8. EMBO reports 2003, 4:p1 (kinase)9. Curr Opin Microbiol 2003, 6:p146 (cell size)10. Genome Biol 2003, 4:p204 (microarray)11. Annu Rev Biochem 2002, 71:p333 (eukaryotes)12. Nature Cell Biology 2004, 6:p648 (origin firing)13. Acta Biochimica Polonica 2005, 52:p1 (SSB)14. GENES VIII chapter 14 (Introduction)

Learning objectives

Let the students know where we are and where to go about DNA replication.

Prokaryotic cells Eukaryotic cells

the same or different ?

“ This structure has novel features which are of considerable biological interest ”

Nature 171, April 25 (1953) p. 737-738 - by James Watson and Francis Crick

Molecular structure of nucleic acid

The principles of DNA replication

template dependent requires the four dNTPs primer-dependentsynthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'

OrganismGenome

size (bases)

Estimated genes

Human (Homo sapiens)* 3200 x 106 20,000-25,000

Laboratory mouse (M. musculus)

2600 x 106 20,000

Mustard weed (A. thaliana)

100 x 106 25,000

Roundworm (C. elegans) 97 x 106 19,000Fruit fly (D. melanogaster) 137 x 106 13,000Yeast (S. cerevisiae) 12.1 x 106 6,000Bacterium (E. coli) 4.6 x 106 3,200Human immunodeficiency virus (HIV)

~9700 9

Hepatitis C virus (HCV) ~9030 1SARS-associated Coronavirus

~29,700 ~11

The central questions to be answered

(1) Does DNA synthesis begin at a defined place ?

(2) What determines replication initiation sites ?

(3) What regulates an origin to fire once and only once per cell cycle ?

FeatureProkaryotic (E. coli)

Eukaryotic (human HeLa cells in culture)

DNA content (bp)

4.6 x 106 3.2 x 109

DNA replication rate (nt/sec/rep/ fork)

850 60-90

Number of replication origins/cell

1 103-104

Hours for complete genome replication

0.67 8

Chronicle for DNA replication

1950 1960 1970 1980 1990 2000

In 1972, Kornberg et al. demonstrated in vitro enzymatic activity of DNA polymerase from E. Coli

J. Biol. Chem. 1972;247:p241.


1950 1960 1970 1980 1990 2000

In 1984, the first mammalian-based DNA replication system (SV40) that initiated DNA synthesis successfully in vitro was developed.Proc Natl Acad Sci U S A. 1984;81:p6973.

In 1985, Greider and Blackburn identified enzymatic activity of the telomerase in Tetrahymena extract Cell 1985;43:p405.


1950 1960 1970 1980 1990 2000

Establish the molecular mechanism of DNA replication, involving the stages of initiation, priming, elongation and termination

Establish the linkage of DNA replication to cell cycle control

Study on DNA replication

1. E. Coli model2. Yeast genetic model3. Simian virus 40 (SV40) model4. Adenovirus model5. Herpes simplex virus (HSV) model

Genetic approach: temperature sensitive(ts) mutants

Biochemical approach: in vitro complementation assay

Priming for DNA replication DNA polymerases is incapable of initiating

DNA polymerization in the absence of an existing 3' -OH

The priming RNA supplies the necessary 3' -OH group to prime DNA polymerization and is later clipped off and replaced with DNA by the DNA polymerase

http://www.mansfield.ohio-state.edu/~sabedon/biol1040.htm#dna

http://www.mansfield.ohio-state.edu/~sabedon/biol1020.htm#polymer

http://www.mansfield.ohio-state.edu/~sabedon/biol1040.htm#RNA

http://www.mansfield.ohio-state.edu/~sabedon/biol1040.htm#dna

Priming for DNA replication

Due to low fidelity of RNA polymerases, primase and primosome takes over for priming RNA synthesis.

Okazaki fragment formation requires repeated initiation of DNA replication de novo.

Three kind of priming: an RNA primer, a nick in DNA, a priming protein

http://www.mansfield.ohio-state.edu/~sabedon/biol1065.htm#rna_polymerase

Priming for DNA replication

RNA primer:The majority of prokaryotic and eukaryotic DNA replication

DNA primer: nick translation, hairpin loop

Protein (terminal protein + dCTP) as a primer:adenovirus DNA replication

energy source

energy source

Why DNA synthesis is 5’ to 3’ ?

Replication complex and replication forkBi-directional DNA replicationEnzymology of DNA replication


The DNA replication fork

Lagging strand with Okazaki fragments (1000-2000 base)

Leading strand

Most recently synthesized DNA

DNA replication fork

Replication complex and replication forkBi-directional DNA replicationEnzymology of DNA replication


Directions of DNA replication

Directions of DNA replication

Experimental evidence of bidirecitonal DNA replication

Large eukaryotic DNA has multiple origins of replication

Replication complex and replication forkWhy DNA synthesis is 5’ to 3’Bi-directional DNA replicationEnzymology of DNA replicationpolymerase, primase, helicase, single strand DNA binding protein (SSB), ligase, topoisomerase, telomerase


Prokaryotic system

Characteristic

Polymerase I

Polymerase II

Polymerase III

gene polA polB polC

MW 103,000 90,000 130,000

#molecules/cell 400 100 103‘ exonuclease

yes yes yes*

5‘ exonuclease

yes no no

Biological function

DNA repair, RNA primer

excision

SOS DNA repair

Replicative chain growth

DNA polymerase of E. coli

*Polymerases IV and V participate in SOS repair function.

Proofreading function

E. Coli DNA Pol III (replicative polymerase)

complex enzyme with ten subunits

subunit contains the active site for nucleotide addition

subunit is a 3’ to 5’ exonuclease, a proofreading activity that removes incorrectly added bases

subunit can stimulate exonuclease

E. Coli DNA Pol III (replicative polymerase)

subunit is a processivity factor -> prevents enzyme from falling off prematurely thus decrease likelihood of frameshift mutation

subunit is a clamp loader -> places the processivity subunit on DNA

subunit is a dimerizing subunit -> link the two catalytic cores together

Note: In mammalian system: PCNA is a processivity factor, RF-C is a clamp loader

Finger Thumb

Palm

DNA

3‘-5’ exonuclease

PDB: 1D8Y

3D structure of DNA polymerase

nucleotideRotate 60

Editing mode Polymerizing mode

3D structure of DNA polymerase

-subunit clamp

subunit

subunit

DNA

waterwater

Assembly of E. Coli DNA Pol III

Clamp loader () cleaves ATP to

load clamp () on DNA

Enter the core enzyme ()

Assembly of E. Coli DNA Pol III

Dimerization by subunits

Repetitively loading on the lagging strand

Stages of DNA replication

Initiation Elongation Termination

Note: At the initiation stage, origin binding proteins (OBP) and ATP is required to trigger the following events • Melt the two strands of DNA• Recruit protein factors essential for replication

Origin for DNA replication in E. coli

245 bp ori C region

Seven replication factors working at the origin-DnaA: origin binding protein (OBP)-DnaB: helicase-DnaC: recognition protein-HU: bend DNA structure-Gyrase: rotate one strand over the other -SSB: single strand binding -DnaG: primase

13 bp sequences DnaA-binding sites (3 repeats) (4 repeats of 9 bp)

3D structure of DnaAATPases Associated with various cellularActivities (AAA+)

DBD

Mg2+ ADP cofactor

Note: Common features are shared in the initiator of chromosomal replication in bacteria, archaea and eukaryotes FEMS Microbiology Review 2003, 26, p533

DnaA

Initiation of DNA replication in E. coli

DnaB

DnaB

DnaBDnaB

DnaG

DnaG

A-T rich region

Origin for DNA replication in E. coli

Prepriming complex Replication bubble

DnaA

DnaB

DnaB

DnaBDnaB

DnaG

DnaG

Dna A

Regulatory inhibition of DnaA

DnaA protein undergoes conformation change in hydrolysis of ATP and unable to initiate further replication rounds

Note: DnaA protein can be reactivated either by acidic phospholipids or DnaK chaperon, that exchange ADP by ATP

DnaB

DnaB

DnaBDnaB

DnaG

DnaG

Competition model for DnaA-ATP and DnaA-

ADP: coupling the initiation of chromosome replication to cell size Current Opinion in Microbiology 2003, 6:p146

eclipse

DNA replication

Cell growth

Total DnaA-ADP (inactive)

Total DnaA-ATP (active)

Old DnaA-ATP (active)

New DnaA-ATP (active)

completion of cell division

The factors that regulate an origin to fire only once per cell cycle

Convert DnaA-ATP to DnaA-ADP Repress DnaA transcription

Prevent DnaA binding -> DNA replication

-> Hemimethylated GATC at the oriC DNA-> sequestered to plasma membrane

-> thus a membrane-bound inhibitor prevents re-initiation of the origin by DnaA

DNA helicase

~ 60 bp

DNA helicase

Single strand binding protein (SSB)


The SSBs from prokaryotes and eukaryotes share a common core ssDNA-binding domain

Application of SSB in biotechnology

• Increase amplification efficiency in single PCR

• Prevention of primer dimer formation in multiplex PCR

• Increased size of cDNA in RT-PCR

Replication complex and replication fork

Leading strand template

Lagging strand template

Leading strand

Okazaki fragment

Dimer of Pol IIIPrimase


Lagging strand synthesis in E. Coli

Characteristic

Polymerase I

Polymerase II

Polymerase III

gene polA polB polC

MW 103,000 90,000 130,000

#molecules/cell 400 100 103‘ exonuclease

yes yes yes*

5‘ exonuclease

yes no no

Biological function

DNA repair, RNA primer

excision

SOS DNA repair

Replicative chain growth

DNA polymerase of E. coli

Note: In mammalian system: RNAse HI and FEN1 remove the RNA primer

The replication termini in E. Coli

Binding of tus protein -> stop DnaB helicase activity

Wake up

Eukaryotic system

Eukaryotic model system

1. Yeast2. Simian virus 40 (SV40)3. Early embryos of Drosophila

and Xenopus 4. Mammalian cells5. Adenovirus

Polymerase Location

Size of catalytic subunit

(kD)

Biological function

Alpha ()/primase

nucleus 160-185Priming &

Lagging strand replication

Delta () nucleus 125Leading strand

replication

Epsilon nucleus210-230 or

125-140

Lagging strand replication & DNA repair

Beta () nucleus 40 DNA repair

Gamma ()mitochondri

a125 Mitochondrial

DNA replication

DNA polymerase of eukaryotic cells High fidelity enzyme

Polymerase LocationBiological function

Zeta () nucleusThymine dimer

bypass

Eta () nucleus Base damage repair

Iota () nucleus Required in meiosis

Kappa () nucleusDeletion & base

substitution

DNA polymerase of eukaryotic cells

Low fidelity enzyme

06_08_monitorprogress.jpg

DNA microarrays can be used to locate DNA replication origins

Species Replication origin

S. cerevisiaeCore A + B1, B2, B3 elements (~50

bp) (autonomously replicating sequence, ARS)

S. pombe spread over 800-1000 bp

Early embryos of Drosophila and Xenopus

Little or no sequence specificity

Drosophila follicle cells

Specific cis-acting elements, not yet defined sequences

Human -globin

Specific cis-acting elements, not yet defined sequencesNote: AT-rich sequences might be required at the

replication origin

Replication origin of eukaryotic cells

Mitochondrial DNA replication starts at a D-loop

Where does DNA replication take place ?

S phase

0 h 5 h 8 h 12 h 15 h

Tracing by (1) biotin-labelled dUTP (2) bromodeoxyuridine (BrdU) (3) GFP-tagged proteins (GFP-PCNA)

C2C12 mouse myoblast cell

J Cell Biol. 2000 Apr 17;149(2):271-80.

Proliferating cell nuclear antigen

Where does DNA replication take place ?

S phase

0 h 5 h 8 h 12 h 15 h

early S-phase mid S-phase late S-phase

Note: DNA replication takes place at replication foci in the Nucleus, where the replication factors are assembled.

J Cell Biol. 2000 Apr 17;149(2):271-80.

nucleoli

Replisome within a replication focus

Features of DNA replication in eukaryotes

Initiation: licensing and activationformation of pre-replication complex (pre-RC) -> initiation of replication complex (RC)

Elongation Termination: telomere and telomerase

Note: The origin binding proteins (OBP), forming origin recognition complex (ORC), are required for assembly of the pre-RC.

Licensing and activation of DNA replication

origin

Licensing factors binding

Note: The DNA replication events of licensing and activation are coordinated with cell cycle progress. BioEssays 2003, 25: p116

M/G1

Early S

molecular combing and fiber spreading studies

Digoxigenin-dUTP is added at t = 0, and biotin-dUTP is added at t = 10, 20, or 30 min after the release

Note: Linear macromolecule, like DNA, which can be anchored on lysine-coating surface by their

extremities only.

10’

20’

30’

early fork

Distribution of eye-to-eye distances (ETED)

Note: the ETED was measured by electron microscopy, molecular combing and fiber spreading studies

Sperm nuclei replicated in Xenopus egg extracts

10 kb

J Mol Biol 2002;320:p741

Licensing and activation of replication origins

BioEssays 2003, 25: p116

Licensing factors:ORC (origin recognition complex) 1-6, CDC6, CDT1

G1/S transition: CDC7 and sCDK activation ----->(1) Loading the DNA polymerase and DNA replication progresses(2) Releasing of MCM2-7

Note: reloading of MCM2-7 is prevented by CDKs and geminin until cells pass through mitosis

G1 phase

S phase

Licensing and activation of replication origins

BioEssays 2003, 25: p116

Note: the ORC binding site and replication origin (AT-rich repeats) locate as neighbors but not the same site

Inactivation of other MCM complexesAT-rich repeats

ORC 1-6, CDC6, CDT1M/G1 phase

S phase

G1 phase MCM2-7

CDC45

ATR and ATM regulate the timing of DNA

replication origin firing Nature cell biology 2004, 6, p648

ATM: Ataxia telangiectasia mutated ATR: Rad3-related-kinase

ATM/ATR feedback

+ caffeine to inhibit ATR/ATM

DNA damage -> activate ATM/ATR

-> down-regulate cdc7/cdk2 -> Inhibit origin firing

ORC cycle in eukaryotic DNA replicationGene 2003, 310:p1

Note: the ORC cycle restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cycle binding site

3D structure of origin binding protein (OBP)

AAA+ superfamily

DnaADnaA CDC6 CDC6

FEMS Microbiology Review 2003, 26, p533

Mg2+ ADP cofactor Mg2+ ADP cofactor

Phylogenetic tree analysis of ORBs Based on amino acid sequences

FEMS Microbiology Review 2003, 26, p533

Simian virus 40 DNA replication in vitro

Li JJ, Kelly TJ.

A biochemical assay for the replication activity -Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with SV40 -Catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. -Extracts prepared from uninfected monkey cells also support replication only in the presence of added SV40 large tumor (T) antigen

-The in vitro replication reaction proceeds via branched intermediates (theta structures).

Proc Natl Acad Sci U S A.

1984;81:p6973.

Re-constitution of purified components of the DNA synthesome

Study of simian virus 40 DNA replication in vitro

replication origin

SV40 T-Ag-Initiator: binds to the origin

-Helicase: unwinds duplex DNA

-RPA: single-stranded DNA binding protein

-DNA polymerase /primase

- Replication factor C (RF-C): clamp loader

protein

-Proliferating cell nuclear antigen (PCNA):

a general recruiting factor/processivity factor

a. displace pol- by Pol- or Pol-

b. recruit Fen 1 (exonuclease) and Lig 1 (ligase)

-Pol : DNA elongation

RNA-DNA primer synthesis

SV40 DNA replication in vitro

-SV40 T-Ag: binds to the origin/unwinds duplex DNA

-RPA: single-stranded DNA binding protein

-DNA polymerase /primase: RNA primer synthesis

-PCNA/RFC: release DNA polymerase

-Pol : DNA elongation

DNA-RNA primer

SV40 T-Ag/helicase


-RNase H: remove RNA primer

-FEN1 (5’-3’ exonuclease): remove RNA primer

-DNA ligase I: joins the end of the Okazaki fragments

DNA-RNA primer

SV40 T-Ag/helicase


Eukaryotic DNA replication synthesome

At least two different DNA polymerases ( and )A single-stranded DNA-binding protein (RPA)A clamp-loading complex (RFC, PCNA)A polymerase clamp combine to replicate DNA

Annu Rev Biochem 1998;67:p721

Core proteins at the DNA replication forkNature, 2003, 421, p431

Eukaryotic DNA replication synthesome

ori

distributive mode: pol processive mode: pol and (RF-C-independent) (RF-C-dependent)

oriPCNA

PCNARF-C

Primosome complex

RF-C

EMBO J 2002;21:p2485

inhibitor to pol

Isolation of replication competent complex from nucleus

Chromatography

Nuclear extract

Protein identification by westernin the high-molecular weight RC complex

EMBO J 2002;21:p2485

Gel filtration

Nuclear extract

Protein identification by western in the high-molecular weight RC complex

Note: RC complex consists of (1) replication factory (pol , pol , RF-C, ligase I, topo I, RP-A)(2) cell cycle regulators

Isolation of replication competent complex from nucleus

EMBO J 2002;21:p2485

Cell cycle progress and checkpoint control

Pol activity Pol activity RC compositionRC composition

EMBO J 2002;21:p2485

CDK-driven replication switch model

Replication factory (replication synthesome)

- pol , pol , RF-C, ligase I, topo I, RP-A

Dynamic of the DNA synthesome

At G1/S transition, DNA synthesome are

maintained as chromatin-free

At S phase, there are two forms of DNA

synthesome

At G2 phase, DNA synthesome are

maintained as chromatin-free EMBO 2003, 4, p666

Phosphorylation of the DNA synthesome

Phosphorylation of Pol-/primerase (early)1. Pre-replication complex

-> replication complex2. Inhibition of the re-initiation of

replication origin

Phosphorylation of ligase I(late)1. Response to DNA damage

DNA replication factory (DNA synthesome)

EMBO 2003, 4, p666

Replication foci

Assorted replication factors

DNA replication DNA repair DNA recombination Chromatin remodelling

- Replication protein A - DNA polymerase-- DNA polymerase- - DNA ligase I- DNA topoisomerase II- - DNA-C5-methyltrasferase- Histone deacetylase - Chromatin assembly factor- MSH3/MSH9 - hMTH- MRE11 - Cdk2/Cyclin A

Chromatin dynamics at DNA replication fork

Eur. J. Biochem 2004, 271, p2335

HAT: histone acetyltransferaseCR: chromatin remodeling machinery

helicase

DNA replication

Telomeres

AGGGTT AGGGTT AGGGTT 3‘

TCCCAA 5‘Newly synthesized tagging strand

Telomere repeats

Telomeres consist of several hundred to a few thousand base pairs composed of repetitive oligomeric sequences with a high G contents (e.g. AGGGTT)

3’ end of G-rich strand extends in single-stranded form 10-16 nucleotides beyond 5’ end

Telomeric sequences differ depending on the organism but are in each case generated by the telomerase enzyme

Telomerase enzyme

telomerase RNA

Note: The telomerase enzyme consists two parts: telomerase reverse transcriptase (TERT)and telomerase RNA (TR)

Cancer letter 2003, 194, p139

Telomerase mechanism of action

Telomerase reverse transcriptase

Telomerase RNA

Note: the G-strand extension, telomere elongation and DNA replication may occur contemporaneously

telomerase RNA

Mutations of telomerase RNA is associated with an autosomal dominant variant of dyskeratosis congenita

Note: Patients exhibit reduced levels of the telomerase RNA and shortened telomere when the 3’ end TR is mutated

Nature 2001;413:p432

(1) The function of telomerase is to compensate for the shortening of telomeres that occurs at each replication cycle. (2) Telomerase is turned off in many somatic cells, typically when differentiation occurs. Continued division in cells that lack telomerase activity will cause the telomeres to shorten in each generation. The cells are not capable to continue propagation once telomere lengths have become too short. (3) When cells enter senescence as the result of telomere shortening, p53 is activated, leading to growth arrest or apoptosis. (4) The telomerase activity usually is reactivated in tumors. The process of immortalization might be involved the reactivation of telomerase.

Telomerase support for continued cell division

Proteins associated with human telomerase activity

TEP1 non-steiochiometric association with activity

Stau binds hTR L22 binds hTR Dyskerin binds hTR P23/p90 binds amino terminus of hTERT hnRNPs associated with activity; affect

telomere length

The central questions to be answered

(1) Does DNA synthesis begin at a defined place ?

(2) What determines replication intiation sites ?

(3) What regulates an origin to fire once and only once per cell cycle ?

The endThe end

Questions

1. How to regulate an origin to fire once and only once per DNA replication cycle (prokaryotes & eukaryotes) ?

2. How the DNA telomerase work ? Describe the biological significance of DNA telomerase in DNA replication.

3. How to coordinate DNA replication and cell cycle in eukaryotes ?

dna replication

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