replication of dna - surendranathcollege.org · 3/26/2020 · v dna replication is a means to...

Replication of DNAReplication of DNA

PHYA- Sem-IVPHYA- Sem-IV Molecular Biology Molecular Biology

Compiled and prepared byCompiled and prepared by Dr Barnali Ray Basu Dr Barnali Ray Basu

Department of Physiology Department of Physiology Surendranath College Surendranath College

3/25/2020 1@Surendranath College

Dr. Barnali Ray Basu

DNA StructureDNA Structure

Double Helix (2 strands of DNA) Complementary strands pair up (A & T, C & G)– hydrogen bonds Strands are antiparallel (5’ and 3’ ends) Helix (2 strands of DNA)



DNA Replication

1. Models of DNA replication: Meselson-Stahl Experiment

2. DNA synthesis and elongation

3. DNA polymerases

4. Origin and initiation of DNA replication

5. Prokaryote/eukaryote models (circular/linear chromosomes)

6. Telomere replication

7. Histone/chromatin assembly



The three models for DNA replicationThere were three basic models for DNA replication that had been proposed by the scientific community after the discovery of DNA's structure. These models are •Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).•Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.•Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.



Models of DNA replicationModels of DNA replication



http://upload.wikimedia.org/wikipedia/commons/a/a2/DNAreplicationModes.png

The models were tested by Meselson and Stahl, who labeled the DNA of bacteria across generations using isotopes of nitrogen(N15).

From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that DNA is replicated semi-conservatively.



Meselson and Stahl Experiment (1957)Meselson and Stahl Experiment (1957)

• Worked with E. coli. • Discovered the mechanisms of DNA synthesis.• Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase (Kornberg enzyme)

4. Mg 2+ (optimizes DNA polymerase activity)



1. Bacteria were grown in a medium containing nitrogen 15 (N15) for several generations

2. If the medium contains no other nitrogen source, the E. coli will use N15 and incorporate it into their DNA

3. Eventually, they will only have N15

4. Once the E. coli had only N15 they were put into a growing medium contain only N14

5. N15 is heavier than N14 making new incorporation of nitrogen easy to distinguish

6. The differences were measured according to the densities of the new strands

Meselson and Stahl Experiment (1957)Meselson and Stahl Experiment (1957)



DNA ReplicationDNA Replication

Replication is semi-conservative (one strand is old, one strand new)



v DNA replication is a means to produce new molecules that have the same base sequence

v Occurs during interphase of the cell cylcev Replication is semi-conservativev The parent DNA strand separates into twov Each strand serves as a template for new

complementary strandsv The new double helix is half original




DNA elongation (Fig. 3.3a):



Three main features of the DNA synthesis reaction:

1. DNA polymerase-I catalyzes formation of phosphodiester bondbetween 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP.

• Energy for this reaction is derived from the release of two of the three phosphates of the dNTP.

2. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process.

• rate ≤ 800 dNTPs/second

• low error rate

3. Direction of synthesis is 5’ to 3’

DNA polymeraseImage -DNA polymerase-I credit:Protein Data Bank



In prokaryotes, there are three main types of DNA polymerase

Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) #Copies

I Yes Yes Yes 400

II Yes Yes No ?

III Yes Yes No 10-20

•3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain

•Important proofreading ability

•Without proofreading error rate (mutation rate) is 1 x 10-6

•With proofreading error rate is 1 x 10-9 (1000-fold decrease)

•5’ to 3’ exonuclease activity functions in DNA replication & repair.



The major replication events in a prokaryotic cell

(A)Nucleoside triphosphates serve as a substrate for DNA polymerase, according to the mechanism shown on the top strand. Each nucleoside triphosphate is made up of three phosphates (represented here by yellow spheres), a deoxyribose sugar (beige rectangle) and one of four bases (differently colored cylinders).

(B)The three phosphates are joined to each other by high-energy bonds, and the cleavage of these bonds during the polymerization reaction releases the free energy needed to drive the incorporation of each nucleotide into the growing DNA chain. The reaction shown on the bottom strand, which would cause DNA chain growth in the 3' to 5' chemical direction, does not occur in nature.



(C) DNA polymerases catalyse chain growth only in the 5' to 3' chemical direction, but both new daughter strands grow at the fork, so a dilemma of the 1960s was how the bottom strand in this diagram was synthesized. The asymmetric nature of the replication fork was recognized by the early 1970s: the leading strand grows continuously, whereas the lagging strand is synthesized by a DNA polymerase through the backstitching mechanism illustrated. Thus, both strands are produced by DNA synthesis in the 5' to 3' direction.

© 2002 From Molecular Biology of the Cell, 4th Edition by Alberts et al.



Replication takes place at several places on one double helix at the same time

Once the double helix is unwound and unzipped the two parent strands become the leading and lagging strands

This happens because the strands are antiparallel


3’

5’ 3’

5’



DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand:

•Unwinding of any single DNA replication fork proceeds in one direction.

• The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’.

Solution: DNA is made in opposite directions on each template.

üLeading strand : • Synthesized 5’ to 3’ in the direction of the replication fork movement.• continuous , requires a single RNA primer

üLagging strand :•Synthesized 5’ to 3’ in the opposite direction.• semidiscontinuous (i.e., not continuous)• requires many RNA primers , DNA is synthesized in short fragments.



Initiation of replication: Major elements:

ü Segments of single-stranded DNA are called template strands.ü Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

ü Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).(Hydrolysis of ATP causes a shape change in DNA helicase)

ü DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis),

ü Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.

ü Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer.

ü The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase.

ü Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process.



Origin of replication (e.g., the prokaryote example):

ü Begins with double-helix denaturing into single-strands thus exposing the bases.

ü Exposes a replication bubble from which replication proceeds in both directions.

~245 bp in E. coli



3

Polymerase III

5’ 3’

Leading strand

base pairs

5’

5’

3’

3’

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase +

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments1

RNA primer replaced by polymerase I& gap is sealed by ligase



DNA replication (the leading strand)

• Replication is continuous• There are no fragments• Helicase unwinds and unzips the double helix• DNA polymerase III adds nucleotides in the

direction of 5’→3’• DNA polymerase only works in the direction of

5’→3’



DNA Replication (the lagging strand)

The lagging strand runs from 3’ to 5’ 1. DNA replication begins there must be and RNA primer 2. The RNA primer is made by adding complimentary RNA

nucleotides to the lagging DNA strand by hydrogen bonding of the bases RNA has uracil instead of thymine

3. RNA primase (an enzyme) then bonds the RNA nucleotides together

3’ 5’RNA primase

RNA primer



3’ 5’

RNA primerDNA polymerase III

Okazaki fragment

4. After RNA primer is in place DNA nucleotides are added by DNA polymerase III

5. Eventually, the segment of DNA will run into another RNA primer

6. The DNA segments are called Okazaki fragments



Model of replication in E. coli





DNA-Polymerase-III

Functional component Subunit Mass (kDa) Gene Activity or function

Core polymerase a 129.9 polC=dnaE 5' to 3' polymerase

e 27.5 dnaQ=mutD 3'-5' exonuclease

q 8.6 Stimulates e exonuclease

Linker protein t 71.1 dnaX Dimerizes cores

Clamp loader g 47.5 dnaX Binds ATP

(or g complex) d 38.7 Binds to b

(ATPase) d' 36.9 Binds to g and b

c 16.6 Binds to SSB

y 15.2 Binds to c and g

Sliding clamp b 40.6 dnaN Processivity factor

Subassemblies of DNA polymerase III, major subunits, genes and functions



DNA replication (a quick review) Lagging strand

Formed in short segment of 100-200 nucleotides (these are the Okazaki fragments)

Grows in direction of 5’→3’ because DNA polymerase III only works in the 5’→3’

1. Helicase unwinds and unzips the parent DNA 2.RNA primer is formed by RNA nucleotides that are joined

together by RNA primase 3. DNA polymerase III bonds DNA nucleotides to the RNA

primer 4. DNA polymerase I replaces the RNA primer with DNA

nucleotides 5. DNA ligase joins the Okazaki fragments together

Leading strand 1. Helicase unwinds and unzips 2. DNA polymerase III adds complimentary DNA nucleotides



D-Loop ReplicationD-Loop Replication

§D-loop replication is a proposed process by which circular DNA like chloroplasts and mitochondria replicate their genetic material. § chloroplasts and mitochondria have a single circular chromosome like bacteria instead of the linear chromosomes found in eukaryotes.



https://en.wikipedia.org/wiki/Chloroplast

https://en.wikipedia.org/wiki/Mitochondria

https://en.wikipedia.org/wiki/Chloroplast

https://en.wikipedia.org/wiki/Mitochondria

https://en.wikipedia.org/wiki/Chromosome

https://en.wikipedia.org/wiki/Bacterium

https://en.wikipedia.org/wiki/Chromosome

https://en.wikipedia.org/wiki/Eukaryote

Dr Barnali Ray Basu

Process of D-loop Process of D-loop replication:replication:The D loop maintains The D loop maintains an opening in an opening in mammalian mammalian mitochondrial DNA, mitochondrial DNA, which has separate which has separate origins for the origins for the replication of each replication of each strand.strand.



replication of dna - surendranathcollege.org · 3/26/2020 · v dna replication is a means to...

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