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LSM1102 - Lec 2a - DNA Replication - Part I

DNA replication is the process by which the genetic material is copied. The original DNA strands are used as templates for the synthesis of new strands. The AT / GC rule or Chargaff's rule ensures that T signals addition of a on new strand, and g signals addition of C.

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101 views

LSM1102 - Lec 2a - DNA Replication - Part I

DNA replication is the process by which the genetic material is copied. The original DNA strands are used as templates for the synthesis of new strands. The AT / GC rule or Chargaff's rule ensures that T signals addition of a on new strand, and g signals addition of C.

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givena2ndchance
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DNA REPLICATION

Adapted from Chapter 11 in Genetics: Analysis and Principles (Robert J. Brooker)

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA replication is the process by which the genetic material is copied


The original DNA strands are used as templates for the synthesis of new strands DNA replication relies on the complementarity of DNA strands
The AT/GC rule or Chargaffs rule

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Double helix unwinds Each strand acts as template Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C Two daughter helices produced after replication

Which Model of DNA Replication is Correct?

Conservative model Both parental strands stay together after DNA replication Semiconservative model The double-stranded DNA contains one parental and one daughter strand following replication Dispersive model Parental and daughter DNA are interspersed in both strands following replication

In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models
They found a way to experimentally distinguish between daughter and parental strands 15N (a heavy isotope of Nitrogen) was used to label parental DNA strands 14N (a light isotope of Nitrogen) was used to label daughter DNA strands

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Nitrogen occurs as 2 isotopes: DNA labeled with labeled with 14N


15N

14N

and 15N.

is heavier than the one

Molecules of different density can be separated by a procedure called equilibrium density-gradient centrifugation. The density of 6M CsCl is ~1.7g/cm3; at equilibrium, the linear density gradient produced by ultra-high speed centrifugation [50,000 rpm (Revolutions Per Minute), 48-72 h] will range from 1.65 g/cm3 at the top of the tube to 1.75 g/cm3 at the bottom of the tube. The densities of most naturally occurring nucleic acids fall within this range.

Figure 11.3

DNA

Figure 11.3

11-10

Where Does DNA Replication Start?


Origin Origin

Unidirectional Bidirectional Circular DNA

Replication is Bidirectional
Origin

Unidirectional

Bidirectional

BACTERIAL DNA REPLICATION


DNA synthesis begins at a site termed the origin of replication Each bacterial chromosome has only one Synthesis of DNA proceeds bidirectionally around the bacterial chromosome The replication forks eventually meet at the opposite side of the bacterial chromosome This ends replication

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Initiation of Replication
The origin of replication in E. coli is termed oriC
origin of Chromosomal replication

Three types of DNA sequences in oriC are functionally significant


AT-rich region DnaA boxes GATC methylation sites Refer to Figure 11.5
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-15

Figure 11.5

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11-16

Figure 11.6

Other proteins such as HU and IHF also bind. This causes the region to wrap around the DnaA proteins and separates the AT-rich region

DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex

11-17

Figure 11.6

Composed of six subunits Travels along the DNA in the 5 to 3 direction Uses energy from ATP

Bidirectional replication

11-18

DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork
DNA gyrase (topoisomerase) travels ahead of the helicase and alleviates these supercoils using energy from ATP

Single-strand binding proteins bind to the separated DNA strands to keep them apart Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase
These short RNA strands start, or prime, DNA synthesis
They are later removed and replaced with DNA

Breaks the hydrogen bonds between the two strands

Keep the parental strands apart

Alleviates supercoiling

Synthesizes an RNA primer

Figure 11.7

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-20

DNA Polymerases
DNA polymerases are the enzymes that catalyze the attachment of nucleotides to make new DNA In E. coli there are five proteins with polymerase activity
DNA pol I, II, III, IV and V DNA pol I and III
Normal replication

DNA pol II, IV and V


DNA repair and replication of damaged DNA
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11-21

DNA Polymerases
DNA pol I
Composed of a single polypeptide Removes the RNA primers and replaces them with DNA

DNA pol III


Composed of 10 different subunits (Table 11.2)
The subunit synthesizes DNA The other 9 fulfill other functions

The complex of all 10 is referred to as the DNA pol III holoenzyme


Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-22

11-23

DNA polymerases
Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA

Figure 11.8
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11-24

Mechanism of Action of DNA Polymerase I


Reaction occurs between the 3-OH group at the end of the primer strand and the interior P atom of the nucleotide being added, with removal of a pyrophosphate (PP). Extension of the strand is always in the 5 > 3 direction.
P

Fig 11.12 Snustad & Simmons

Fig. 11.10 Brooker

DNA polymerases cannot initiate DNA synthesis

Problem is overcome by the RNA primers synthesized by primase Problem is overcome by synthesizing the 3 to 5 strands in small fragments (each fragment: 5 to 3)

DNA polymerases can attach nucleotides only in the 5 to 3 direction

Figure 11.9

Unusual features of DNA polymerase function

11-25

The two new daughter strands are synthesized in different ways Leading strand (continuous)
One RNA primer is made at the origin DNA pol III attaches nucleotides in a 5 to 3 direction as it slides toward the opening of the replication fork

Lagging strand
Synthesis is also in the 5 to 3 direction
However it occurs away from the replication fork

Many RNA primers are required DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each)
These are termed Okazaki fragments after their discoverers
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11-26

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