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Replication/DNA Replication - Lecture Slides | PCB 4522, Study notes of Molecular biology

University of Florida (UF)Molecular biology

Material Type: Notes; Class: MOLECULAR GENETICS; Subject: PROCESS BIOLOGY (CELL/MOLECULAR/ECOLOGY/GENETICS/PHYSIOLOGY); University: University of Florida; Term: Spring 2004;

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Molecular Genetics
PCB4522 Spring 2004
Lecture 2- Replication
Dr. Eva Czarnecka-Verner
Course web page:
http://PCB4522.IFAS.UFL.EDU
--Or go to Microbiology & Cell Science home page
and look under course material.
Chapter 13: DNA Replication
(Chapter 15 in Gene VI &14 in VIII)
Primosome: a protein complex that
initiates synthesis of a DNA strand.
Replisome: complex of proteins
engaged in elongation of the newly
synthesized DNA strand.
Assembles at the replication fork.
Identification of protein components
involved in DNA synthesis
1. temperature sensitive mutants:
conditional lethal mutants; replication at permissive
conditions but fail to function at nonpermissive
conditions (high temp.; 42°C).
In E. coli identified loci- dna genes
2. dna genes:
a. quick-stop mutants: immediate stop in replication;
elongation enzymes defective & defects in precursors.
b. slow-stop mutants: defective in reinitiation (smaller
class).
Identification of protein components
involved in DNA synthesis
3. in vitro complementation systems :
combine extracts from mutant and
wild-type strains. Can add back
purified proteins to identify function
of a specific dna gene product.
Progress much slower in eukaryotes.
DNA polymerases: enzymes that make DNA
1. Both bacteria and eukaryotes contain multiple DNA
polymerases.
2. The ones that actually replicate the DNA are called
“DNA replicases.”
3. All have the same type of synthetic activity:
a) each can extend a DNA chain by adding
nucleotides one at a time to a 3’ OH end
b) the choice of dNTPs dictated by base pairing
with the template strand
DNA polymerases: enzymes that make DNA
5. Bacterial DNA replicases contain a large
number of subunits (large protein assemblies).
It is hard to say which proteins are actually
subunits and which proteins are just loosely
associated.
4. Some function as independent enzymes
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Molecular Genetics

PCB4522 Spring 2004

Lecture 2- Replication

Dr. Eva Czarnecka-Verner

Course web page:

http://PCB4522.IFAS.UFL.EDU

--Or go to Microbiology & Cell Science home page and look under course material.

Chapter 13: DNA Replication

(Chapter 15 in Gene VI &14 in VIII)

Primosome: a protein complex that

initiates synthesis of a DNA strand.

Replisome: complex of proteins

engaged in elongation of the newly

synthesized DNA strand.

Assembles at the replication fork.

Identification of protein components

involved in DNA synthesis

1. temperature sensitive mutants:
conditional lethal mutants; replication at permissive
conditions but fail to function at nonpermissive
conditions (high temp.; 42°C).
In E. coli identified loci - dna genes
2. dna genes:
a. quick-stop mutants: immediate stop in replication;
elongation enzymes defective & defects in precursors.
b. slow-stop mutants: defective in reinitiation (smaller
class).

Identification of protein components

involved in DNA synthesis

3. in vitro complementation systems :

combine extracts from mutant and

wild-type strains. Can add back

purified proteins to identify function

of a specific dna gene product.

Progress much slower in eukaryotes.

DNA polymerases: enzymes that make DNA

1. Both bacteria and eukaryotes contain multiple DNA
polymerases.
2. The ones that actually replicate the DNA are called
“DNA replicases.”
3. All have the same type of synthetic activity:
a) each can extend a DNA chain by adding
nucleotides one at a time to a 3’ OH end
b) the choice of dNTPs dictated by base pairing
with the template strand

DNA polymerases: enzymes that make DNA

5. Bacterial DNA replicases contain a large

number of subunits (large protein assemblies).

It is hard to say which proteins are actually

subunits and which proteins are just loosely

associated.

4. Some function as independent enzymes

1. pol I: (encoded by polA gene)

a) Major repair enzyme for damaged DNA
b) Plays secondary role in semiconservative
replication.
c) Most abundant (400/cell)
d) Molecular mass of 103 kD

Five DNA polymerases in E. coli

2. pol II: (encoded by polB gene)

a) Minor DNA repair enzyme
b) Molecular mass of 90 kDa
3. pol III: (encoded by polC/dnaE gene):
a) REPLICASE; de novo synthesis of
new strands of DNA
b) Contains many subunits
c) There are 10-20/cell
c) Molecular mass of 900 kDa
c) Has no 5’ to 3’ exonuclease activity

Five DNA polymerases in E. coli

4. pol IV: (encoded by dinB gene)

a) SOS repair enzyme of damaged DNA

Five DNA polymerases in E. coli

5. pol V: (encoded by umuD’ 2 C gene)
a) SOS repair enzyme of damaged DNA

1. T4, T5, T7 & SPO1:

a) 5’- 3’ synthetic activities
b) 3’- 5’ exonuclease proofreading activities

Phage coded DNA polymerases

2. Mutations prevent phage development
3. Each phage polymerase peptide associates with
other proteins (phage or host) to make the intact
enzyme

Eukaryotic DNA polymerases

Five identified in mammals.
Enzyme α^ (I)

relative activity

δ (III) ε (II) β γ

Location

function

Nuclear

priming of both strands

Nuclear

elongation of both strands

Nuclear

repair & replicati on

Nuclear

repair

Mitochondrial

replication

exonuc.

No Yes Yes No Yes

PRIMASE REPLICASE

Eukaryotic DNA polymerases: PRIMASE

DNA Pol α(Ι):primase complex- bifunctional
Heterotetrameric phosphoprotein
58 kDa protein- tethers primase to 180 kDa subunit
48 kDa PRIMASE- initiates DNA synthesis (makes
complementary RNA primer)
180 kDa polymerase A subunit- extends RNA
primer by making a short DNA (only in Drosophila has

proofreading activity

70 kDa subunit- no known catalytic function
( may recruit polα:primase to the replication fork)

Klenow fragment (DNA pol I)

Catalytic domain of T7 DNA Polymerase

Right hand structure= synthetic domain has 3 parts

Thumb

Fingers Palm

Proofreading exo 3’-5’

dNTP

Large cleft

B DNA

distorted A DNA

60 o

Inward rotation

8 o

Small fragment: has 5’ to 3’ exonuclease only.

This activity allows pol I (intact) to be used for

nick translation in vitro.

DNA pol I ( polA )

N C

Klenow fragment

exonuclease 3’-5’

polymerase

small fragment

exonuclease 5’-3’ Proofreading domain

68 kDa 35 kDa

Note : removes RNA primer & ~10 bp DNA

2. Nick translation: initiates at nicks in

DNA. Extends the 3’ OH end while

removing the strand in front by its 5’-3’

exonuclease activity. Displaces existing

strand.

3. DNA pol I plus DNase is used for in vitro

labeling of DNA by nick translation.

DNA pol I ( polA )

Nick translation by intact

DNA pol I

excised DNA fragments

32 P
5’ -P
3’ -OH

1. add DNase to nick DNA

Add one 32 P-dNTP, three cold dNTPs and DNA pol I

note: nick moves 5’ to 3’

E. coli DNA pol I

in vivo function:

Filling in short stretches of single-stranded

DNA that arise from:

a. DNA replication (lagging strand).

b. DNA repair when damaged bases have

been removed.

E. coli DNA pol III (Replicase)

1. In order to study must use polA mutant strain of E.
coli since the pol I concentration is so great that it
overwhelms pol III activity. In vitro studies use
extracts from polA mutant cells.
2. Subunit structure:
a. α subunit: 130 kDa; DNA synthetic activity; dnaE.
Mutation is lethal.
b. ε subunit: 3’ to 5’ exonucleolytic activity;
proofreading function; dnaQ. Mutations increase error
rate by 10 3.

Initiation of DNA synthesis

All DNA polymerases require a primer to

provide a free 3’-OH end to initiate DNA

synthesis.

Types of priming reactions:

  1. Primase synthesizes a short RNA primer that is then extended by DNA polymerase. (cellular DNA, papova virus)
  2. Extension of the 3’ end of DNA at a nick. (rolling circle replication of ΦΧ 174 )
  3. Protein-dNTP primes directly by presenting first dNTP (adenovirus, bacteriophage)
  4. Pre-existing cellular RNA (mitochondrial genome, retrovirus)

ColE replicon requires a long RNA primer

(Fgs. 12.34, 12.36, 12.37, Genes VII)

1. Transcription- 555 bp RNA primer upstream from **origin of replication, passes origin; has three hairpins

  1. RNase H cleaves RNA primer at origin- free 3’ OH
  2. Persistent (-265; -20) RNA-DNA hybrid remains
  3. DNA synthesis starts= replication
  4. Control:** a) RNA primer precursor is a positive regulator; b) antisense RNA I (108 b) is a negative regulator c) Rom protein enhances RNA I/RNA primer binding **what inhibits replication- transcription continues
  5. Mutations in RNA I and RNA primer pairing region** make mutant plasmid different compatibility group

DNA synthesis is semidiscontinuous and

primed by RNA

The problem: DNA synthesis must always
proceed from 5’ to 3’. As the replication fork
moves, one of the template strands continuously
exposes new “upstream” template.

lagging strand leading strand 5’

leading strand: synthesis can proceed continuously
in the 5’ to 3’ direction.
lagging strand: synthesized in the reverse direction
as a series of fragments which are later joined.
Discontinuous synthesis.

lagging strand leading strand 5’

Semidiscontinuous replication:

The lagging strand fragments are known as

“Okazaki fragments.” Usually 1,000 to

2,000 bases in length.

lagging strand

Semidiscontinuous replication:

RNA primer (~11-12 bases)

(RNA polymerase= dnaG primase)

5’-CTG-3”

GAppp-5’ 1 2

There are two types of DNA replication in E. coli :

1- ΦΧ 174 phage

2- OriC origin of bacterial chromosomal replication:

There are two types of DNA replication in E. coli : 1- ΦΧ174 phage: each strand synthesized separately (unidirectional replication fork)

a) synthesis of the (-) strand to form the double-stranded RF form serves as a model for lagging strand synthesis.

b) synthesis of the (+) strand to form single-strands for packaging into phage particles servers as a model for leading strand sysnthesis.

2- OriC origin of bacterial chromosomal replication: both strands synthesized at the same time (bidirectional replication fork)

ΦΧ174 phage as a simple model for replication:

  • strand (^) Replicative form (RF): ds plasmid

Rolling circle replication

“Lagging strand” synthesis

  • strand packaged to form virion Rolling circle replication is a model for leading strand synthesis.

Two kinds of activities are needed to convert double- stranded RF DNA to single-stranded DNA without synthesis of new DNA. a. Helicase: separates the strands using ATP to provide the energy. b. single-strand binding protein. (SSB).

ΦΧ174 phage replication provides a model for DNA replication

RF

(randomly nicked) (^) (+) (^) SSB = = Rep (helicase)

Note: no DNA polymerase just to separate single strands

Background for Rolling circle replication:

ΦΧ174 as a simple model for replication

Proteins needed for rolling circle replication: a. “gene A” protein to nick at origin (pas). Covalently linked to 5’end of the displaced strand. b. SSB protein to keep DNA single-stranded. Binding is highly cooperative. c. Rep protein provides helicase function. d. DNA pol III holoenzyme

(+)

(+)

Rolling circle replication: Gene A protein

(-)

(+)

SSB

Rep

DNA pol III elongates 3’-end of the nick.