Part Two of BMB 400

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Part Two of BMB 400 needed for DNA replication: Chapter 5 asses Sept 23, 25, Oct 02, 07 vered entire chapter terminators and control of replication: Chapter 6 asses Oct 04, 07. Note that supercoiling is from Chapter 2, pages 7 (questions 2.12-2.14) ver all but “Cellular control of replication” pages 322-326, questi 18, 6.19 and Repair: Chapter 7 ass: Oct 09 strict coverage of mutagenesis to types of mutations and UV damage. t cover these topics on pages 338-348 Errors in Replication Chemical modification by oxidation Chemical modification by alkylation Chemicals that cause deletions Ionizing radiation ver all repair mechanisms t cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16. estions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7 damaged DNA to be repaired.]

description

Part Two of BMB 400. Enzymes needed for DNA replication: Chapter 5 Classes Sept 23, 25, Oct 02, 07 Covered entire chapter Origins, terminators and control of replication: Chapter 6 Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80 (questions 2.12-2.14) - PowerPoint PPT Presentation

Transcript of Part Two of BMB 400

Page 1: Part Two of BMB 400

Part Two of BMB 400

Enzymes needed for DNA replication: Chapter 5Classes Sept 23, 25, Oct 02, 07Covered entire chapter

Origins, terminators and control of replication: Chapter 6Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80

(questions 2.12-2.14)Cover all but “Cellular control of replication” pages 322-326, question 6.11, 6.18, 6.19

Mutation and Repair: Chapter 7Class: Oct 09Restrict coverage of mutagenesis to types of mutations and UV damage.Not cover these topics on pages 338-348

Errors in ReplicationChemical modification by oxidationChemical modification by alkylationChemicals that cause deletionsIonizing radiation

Cover all repair mechanismsNot cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16.

[For questions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7.16 to see the damaged DNA to be repaired.]

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Rest of Part Two

Recombination: Chapter 8Classes: October 11, 16

Transposition: Chapter 9Class October 18

Exam: October 21

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October 07 class

• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome

• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:

2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling

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Primase

• Synthesizes short oligonucleotides from which DNA polymerases can begin synthesis.– Combination of ribonucleotides and

deoxyribonucleotides

• Does not itself require a primer.

• E. coli primase is DnaG, 60 kDa

• Acts within a large primosome.

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Primers made by DnaG

• Primers can be as short as 6 nt, as long as 60 nt.

• Can substitute dNTPs for rNTPs in all except 1st and 2nd positions– Make hybrid primers with dNMPs and rNMPs

interspersed.

• Primase binds to CTG– T serves as template for 1st nucleotide of

primer.

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Primosome has many proteins

Protein gene functionPriA priA helicase, 3' to 5' movement, site recognitionPriB priBPriC priCDnaT dnaT needed to add DnaB-DnaC complex to preprimosomeDnaC dnaC forms complex with DnaBDnaB dnaB helicase, 5' to 3' movement, is a hexamer

DNA dependent ATPase.

Pre-priming complex:

Primase = DnaG

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Assay for assembly and migration of the primosome

+ +-

ss RF

Convert single stranded (ss) X174 to duplex, replicative form (RF)

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Steps in priming and synthesis

PriA

PriBPriCDnaT

DnaCDnaB

primer assembly site

ADP + Pi

ATP

ATP

ADP + Pipreprimingcomplex

SS DNAcoated with SSB

recognition step

primosomeNTPs

PPi's

primer synthesis

DnaG

(primase)

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Activities of DnaB and PriA in replisome

“Sewing machine” model

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Control of DNA replication

Replicon

Origins and terminators

Solutions to the “end problem” (telomeres)

Cellular control mechanisms

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Oct 07 Class

• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome

• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:

2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling

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Replicon = unit that controls replication

InitiatorReplicator Replicator

Initiator

+

E. coli ori C DnaA

Yeast ARS ,

an autonomously

replicating sequence

ORC (the

origin recognition complex)

+ ABF1 ( ARS binding factor 1)

Complex of

Replicator +

initiator allows

replication to

begin

duplex

DNA

Replicator: cis-acting DNA sequence required for initiation; defined genetically

Origin: site at which DNA replication initiates; defined biochemically

Initiator: protein needed for initiation, acts in trans

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Theta-form replication

intermediates visualized in

EM for polyoma virus

B. Hirt

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Labeling of completed DNA molecules can map replication origins

Dana and Natahans, 1972, PNAS: map the replication origin of SV40 by labeling replicating molecules for increasing periods of time, isolating complete molecules, digesting with Hind restriction endonucleases, and determining which fragments havethe most radioactivity.

A

C

D

E

K

FJ

GB

I

H

Physical map of the SV40 DNA fragmentsproduced by cleavage with H. influenzarestriction endonucleases

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2-D gels: map number & position of replication origins1st dimension separates by size

2nd dimensionalsoseparates by shape.

unitlength

twice unitlength

"Y-arc" "Bubble-arc" Related to distance from ori to end of fragment.

Simple Y Bubble Double Y Asymmetric

Fragmentsize doublesduringreplication.

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Positions of oriC and ter in E. coliForks meet and terminate in this approx. 100 kb region

and are 23 bp binding sites for Tus, a "contra-helicase."terC and terB

block progress of Fork 2

terD and terAblock progress of Fork 1

E. coli chromosome

oriC

Replication fork 1

245 bp

Replication fork 2

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Structure of oriC

• 245 bp long– 4 copies of a 9 bp repeat– 3 copies of a 13 bp repeat– 11 GATC motifs

13 1313 9 9 99

1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA121 AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA TCTCTTATTA GGATCGCACT181 GCCCTGTGGA TAACAAGGAT CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT241 GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA GAATGAGGGG TTATACACAA301 CTCAAAAACT GAACAACAGT TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC361 AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT TGAGTAAATT AACCCACGAT

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Initiation at oriC: Model

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Termination and

resolution

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Regulation of replication by methylation

G A T C

C T A G

m

m

G A T C

C T A G

m

G A T C

C T A Gm

G A T C

C T A G

m

m

G A T C

C T A G

m

m

replicate methylate(lags behind replication)

Fully methylated Hemimethylated

dam methylase

Fully methylated

Will replicate Will not replicate Will replicate

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Oct 07 Class

• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome

• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:

2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling