2. Replikasi of DNA

transcript

Replication of DNA

Outline

Basic Features of DNA Replication In Vivo

DNA Replication in Prokaryotes

Unique Aspects of Eukaryotic DNA Replication

Basic Features of DNA Replication In Vivo

DNA replication occurs semiconservatively, is initiated at unique origins, and usually proceeds bidirectionally from each origin of replication.

Synthesis of DNA (RNA,proteins):

1-initiation, 2-extension/elongation, 3-termiantion.

DNA polymerase (protein-enzyme)-essential for conservation of any species 3,000/30,000 nucleotides per minutes

One mistake per billion of nucleotides

DNA Replication is Semiconservative

Each strand serves as a template

Complementary base pairing determines the sequence of the new strand

Each strand of the parental helix is conserved

Semiconservative=half conserve

Possible Models ofDNA Replication

The Meselson-Stahl Experiment:DNA Replication in E. coli is Semiconservative

Bacteria growing with 15N for several generations

Change medium and add 14N

--one generation--two generations--three generations

The Origin of Replication in E. coli

Replicon: is a sequence of DNA at which DNA replication is initiated on a chromosome, plasmid or virus.

-OriC (245 bp)

-AT-rich region (replication bubble)

-13-mer and 9-mer tandem

Mer=repeating unit=parts

Eukaryotic: ARS(Autonomously Replicating sequences)AT-rich region 11bp

N: any nucleotide

Bidirectional Replication of the Circular E. coli Chromosome

-Circular DNA (double strand DNA)--Unwind (access and single strand DNA)--Simultaneous semiconservative replication--Swivel (point of break) Topoisomerases--Y-shape structure=replication fork

Topoisomerases: are enzymes that regulate the overwinding or underwinding of DNA.

Bidirectional Replication: The Phage Chromosome

-Small bacterial virus

-Single stranded DNA (12 bp)

-Cohesive/sticky and complementary ends

-DNA ligase (replication, repair and recombination)

Linear

Circular

replication

• DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix unwind and separate, each serves as a template for the synthesis of a new complementary strand.

• The hydrogen-bonding potentials of the bases in the template strands specify complementary base sequences in the nascent DNA strands.

• Replication is initiated at unique origins and usually proceeds bidirectionally from each origin.

DNA Replication in Prokaryotes

DNA replication is a complex process, requiring the concerted action of a large number of proteins

DNA Polymerases and DNA Synthesis In Vitro

Much of what we know about DNA synthesis was deduced from in vitro studies.

DNA Polymerase I Single polypeptides

5’ to 3’

Triphosphate [dATP]

Free 3’OH group of the DNA strands

Continuous vs discontinuous--leading and lagging strands

Replicating fork

Bacteriophage T4

Continuous vs discontinuous--leading and lagging strands

Replicating fork

Prepriming at oriC in E. coli

--Replication bubbles

Self aggregation

DNA helicase: it separates two annealed nucleic acid Strands.

RNA Primers are Used to Initiate DNA Synthesis

DNA primase: short RNA primerRNA/DNA hybrid(unstable ?)

Perfect conditions for DNA polymerases to work(free 3’OH)

DNA Polymerase I:5'3' Polymerase Activity

DNA Polymerase I:5'3' Exonuclease Activity

DNA Polymerase I:3'5' Exonuclease Activity

DNA Helicase Unwinds the Parental Double Helix

One of the most important event during DNA replication

Single-Strand DNA Binding (SSB) Protein

Access to DNA polymerase

Supercoiling of Unwound DNA

DNA Topoisomerases I: produce single transient breaks of DNA and remove supercoiling

It blocks DNA replication

DNA Topoisomerases II: produce double transient breaks of DNA and negative supercoiling (DNA gyrase)

DNA Topoisomerase I Produces Single Strand

Breaks in DNA

Requirements of DNA Polymerases

Primer DNA with free 3'-OH

Template DNA to specify the sequence of the new strand

Substrates: dNTPs

Reaction: nucleophilic attack

DNA Polymerase III is the True DNA Replicase of E. coli

DNA Polymerase III:--a 900 KDa multimeric protein--Dimers--Holoenzymes

--High fidelity (error ~1 in a 1 x 1012)

Proofreading mechanism

Subunits----Prokaryotes

Subunits----Eukaryotes

The Replication Apparatus in E. coli

Primosome:Initiation of Okazaki fragment during lagging strand

DNA primase and DNA helicase

DnaB and C proteins

Require ATP

DNA helicase:unwinds DNA

DNA primase: synthesis of RNA

Topoisomerase: transient DNA breaks

DNA polymerase III: extend the RNA primers (deoyxribonucleotide). It is holoenzymes

DNA Replication

Synthesis of the leading strand is continuous.

Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.

The E. coli Replisome

Replisome: complete replication apparatus

Rolling-Circle Replication

Replication’s Models-

O-shape

Eye-shape

Y-shape

Rolling-circle (viruses, bacteria , amphibians)

_______________________________________

1- Nick by specific endonucleases

2-parental DNA is intact and functions as template

3-DNA polymerase 5’ to 3’

4- displacement of one of the DNA strand

• DNA replication is complex, requiring the participation of a large number of proteins.

• DNA synthesis is continuous on the progeny strand that is being extended in the overall 5'3' direction, but is discontinuous on the strand growing in the overall 3'5' direction.

• New DNA chains are initiated by short RNA primers synthesized by DNA primase.

• DNA synthesis is catalyzed by enzymes called DNA polymerases.

• All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied.

• All DNA polymerases have an absolute requirement for a free 3’-OH on the primer strand, and all DNA synthesis occurs in the 5’ to 3’ direction.

• The 3’ to 5’ exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired (match) nucleotides at the 3’ termini of primer strands.

• The enzymes and DNA-binding proteins involved in replication assembled into a replisome at each replication fork and act in concert as the fork moves along the parental DNA molecule.

Unique Aspects of Eukaryotic Chromosome Replication

Although the main features of DNA replication are the same

in all organisms, some processes occur only in

eukaryotes.

DNA Replication in EukaryotesShorter RNA primers and Okazaki fragments

DNA replication only during S phase(bacteria will duplicate DNA only in a rich environment)

Multiple origins of replication(bacteria shows one origins of replication)

Nucleosomes(nucleosomes are not present in bacteria)

Telomeres(telomeres are not present in bacteria)

Cell Cycle

--check points----S phase----Mitosis

Replicon: segment of DNAcontaining one Origin (O) andTwo termini (T)

The Eukaryotic Replisome

SV40 virus: DNA virus (histones)

Bacteria replication

--unwind parental DNA (without histones)

----DNA helicase

----Topoisomerase

----Single -strand DNA binding protein

----DNA polymerase III

Eukaryotic Replication Proteins

Eukaryotic replication----parental DNA (with histones)

----Polymerases ()

-------Pol initiation of replication (origins) priming of Okazaki fragment complex with DNA primase

-------Pol synthesis of lagging strand

Pol synthesis of leading strand----accessories proteins: PCNA and Rf-C (sliding clamp)

----Pol have exonuclease activity ( 3’to 5”)=proofreading

----Other Pols (pie, lambda, phi, rho, and mu) do not have exonuclease activity ( 5’to 3”)

----Ribonulceases H1 and FEN-1

Produce the RNA/DNA chain

Proliferating Cell Nuclear Antigen: PCNA

Nucleosome Spacing in Replicating Chromatin

Assembly and disassembly of nucleosomes

Chromatin can have alternative states

Inactive--DNA/histones

Active--Polymerase/TFs

Polymaerase/TFsNO TRANSCRIPTION

HISTONES TRANSCRIPTION

“The addition of either TFs or nucleosomes may form stable structures that can not be changed by modifying the equilibrium with free components”

How is the chromatin structure regulated?

Chromatin remodeling

The Telomere “extension” Problem

DNA polymerase can not replicate the terminal DNA---too big ---not enough space ( 3’-OH, primer)

Telomerase(Reverse Transcriptase)

G-rich telomere sequence5’ to 3’

Aging (early aging….progerias)

Immortality:Cancer and Normal cells

Senescence:normal diploid cells cease to divide, (about 45 to 50 cell divisions).

Telomere Length and Aging Shorter telomeres are

associated with cellular senescence and death.

Diseases causing premature aging are associated with short telomeres.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Apoptosis (self-destruction):programmed cell death

Telomeres Are Essential for Survival

Figure 28.32

Dna polymerases classification as follows:

Prokaryotic DNA polymerasesPol I to V

Eukaryotic DNA polymerases

Pol theta, pie, lambda, phi, rho, and mu.

Based on sequence homology

A, B, C, D, X, Y, and RT

bacterial

Since the parental double helix must rotate 360° to unwind each gyre of the helix, during the semi-conservative replication of the bacterial chromosome, some kind of “swivel” must exist. What do geneticists now know that the required swivel is?

a) Topoisomeraseb) Helicasec) A transient single-strand break produced by the action of topoisomerasesd) A transient single-strand break produced by the action of helicasese) A transient single-strand break produced by the action of Ligase

In the E. coli chromosome the origin of replication, called oriC, is characterized as being rich in:

a) A-G base pairsb) A-C base pairsc) C-G base pairsd) C-T base pairse) None of the above

2. Replikasi of DNA

Documents