Contact Prof. Sive

sive@wi.mit.edu

Put “7.013 student in

subject line!

Office hours

Mon, Fri after class, outside 10-250

Tue 11-12 Whitehead Inst. 401C

or by arrangement

7.013

2.28.07

Molecular Biology I

(DNA replication)

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How-to 1How-to 1 FO

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How-to 2How-to 2

SYSTEMSSYSTEMS

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MS

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MS

BIOCHEM GENETICS CELL BIO.MOL. BIO

STEM

CELLS,

CLONING

REC. DNA

CELL TYPE

3DSTRUCTURE

VIRUSES

CANCER

HUMAN

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LIFELIFE

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BIOLOGY

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Review

Base pairing in DNA

H-bond

Each base-

pair: one

purine and

one

pyrimidine;

same overall

shape

1

Purves 11.6:

Base pairing and uniform shape

of base pairs

leads to formation

of the DNA double helix

2

“prime” = #’s on

the d-ribose

Purves 11.7. Antiparallel, complementary DNA strands

3

1. Principles

double-stranded

DNA

5’3’

3’5’

replication of

complementary strands

Replication of identical DNA molecules

strand-separation

templatetemplate

5’3’

3’5’

H. Sive MIT 2007

4

5’3’

3’5’

double-stranded

DNA

Replication of identical DNA molecules

GATTACA

CTAATGT

GATTACA

strand-separation

CTAATGT

templatetemplate

H. Sive MIT 2007

5

replication of

complementary strands

GATTACA CTAATGT

CTAATGT GATTACA

DNA replication animation/ oversimplification!

6

5’3’

3’5’

double-stranded

DNA

strand- separation, replication from each 3’ end

Generation of identical DNA molecules

by replication of complementary strands

5’

5’

3’

3’

H. Sive MIT 2007

7 See Purves 11.10

Purves 11.10: DNA polymerization:

new nucleotides are added to the 3’OH

8

2. Practice

origins of replication

ori ori

9

+H. Sive MIT 2007

Newly replicated DNA

Bidirectional synthesis

Parental ds DNA

Replicated ds DNA

binds specific proteins

which activate

strand separation

and replication

2. Practice

enzymes

5’3’

3’5’

primer removal5’3’

3’5’ H. Sive MIT 2007

10

5’ 3’RNA primer

synthesisPrimase

Only 1 strand

shown

See Purves 11.14

strand-separation Helicase

Single-stranded

DNA binding

protein

3’polymerization

5’ 3’ DNA polymerasePurves 11.13

11

Purves 4.9

A big problem:

how to rapidly replicate >108 bp per chromosome12

2. Practice

continuous/discontinous

DNA synthesis

ori = origin of replication

5’3’

3’5’

ori

= primer

5’

3’

5’

3’

H. Sive MIT 2007

13

3’

5’

leading strand

lagging strand

leading strand te

mplate

lagging strand template

Continuous

DNA synthesis

Discontinous

DNA synthesis

See Purves 11.6

3’

5’

leading strand

lagging strand

Continuous

DNA synthesis

Discontinous

DNA synthesis

H. Sive MIT 2007

See Purves 11.6

Primer removal by RNAase,

DNA fragments act as primers to

“fill in” gaps and ligase

joins fragments

leading strand

lagging strand

3’

5’

14

DNA replication animation

15

3. DNA repair

will be completed on 3.2.07!!

5’3’

3’5’

double-stranded

DNA

Replication of identical DNA molecules

GATTACA

CTAATGT

GATTACA

strand-separation

CTAATGT

templatetemplate

H. Sive MIT 2007

replication of

complementary strands

GATTACA CTAATGT

CTAATGT GATTACA

5’3’

3’

5’

double-stranded

DNAGATTACA

CTAATGT

GATTACA

strand-separation (one strand shown)

template

H. Sive MIT 2007

16

Outcome of

incorrect replication

incorrect replication

round 1

GATTACA 3’3’ CGAATGT

3’

GATTACA 3’CGAATGT3’

strand-separation

replication round 2

GCTTACA 3’3’ CTAATGT

mutant DNA molecule wild type DNA molecule

5’3’

3’

5’

double-stranded

DNAGATTACA

CTAATGT

GATTACA

strand-separation

template

H. Sive MIT 2007

17

replication of

complementary strand

requires “base pairing site” on DNAPol

GATTACA

ATAATGT

incorrect base!!!

5’3’

3’5’

removed by

3’ to 5’ exonuclease

“proofreading site” on DNAPol

GATTACA

CTAATGTDNA polymerase tries again

correct base!!

Proofreading

TAATGT3’

See Purves 11.19

5’3’

3’

5’

double-stranded

DNAGATTACA

CTAATGT

G TTACA

strand-separation

template

H. Sive MIT 2007

18

incorrect base incorporated,

makes a “bubble”, without H bonds

GATTACA 3’

incorrect removed

correct base filled in

GATTACA

Mismatch repair

3’ C AATGT

A

G

3’ C AATGT

CTAATGT

3’

3’3’

How do repair enzymes know

which strand is correct?

Parental “A/C”s are methylated.

MeMeMe

MeMeMe

MeMeMe

MeMeMe

See Purves 11.19

Protective suits for people with Xeroderma

Nucleotide excision repair

of damaged DNA

(Xeroderma defect)

caused by UV light!!

19

See Purves 11.19