Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 11 March

© R. Paselk 2006
 
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DNA Replication

DNA replication is:

DNA Polymerases

All known DNA polymerases, both from prokaryotes and eukaryotes, share a common characteristics:

E. coli Polymerases

Review DNA info discussed last time as exemplified in text Figures:

There are three DNA polymerases in E. coli. Some of the properties of these enzymes are summarized in the table (text Table 25-1, p 982):

E. coli DNA Polymerase Properties

Pol I Pol II Pol III (core)
Mass (kD) 103 90 130 (sigma), 27.5 (epsilon), 8.6 (theta)
Molecules/cell 400 ? 10-20
Turnover number* 600 30 1200-9000
Processivity

20

40

10-15 (holoenzyme >5,000)
Gene polA polB polC, dnaQ, holE
Polymerization 5'right arrow 3' 5'right arrow 3' 5'right arrow 3'
Exonuclease 3'right arrow 5' & 5'right arrow 3' 3'right arrow 5' 3'right arrow 5'
* nucleotides polymerized/min/active site @ 37° C

DNA pol I: This is the first polymerase discovered and characterized. It serves as a model for the others because it is well understood. Pol I has three active sites on a single peptide chain. The protein, with a single 928 residue peptide chain, can be cleaved into two parts, a 323 residue smaller fragment which carries the 5'-3' exonuclease activity and a larger 605 residue piece known as the Klenow fragment (text Figure 25-8, p 982) which carries the polymerase and 3'-5' exonuclease activity in a single cleft with two widely separated active sites.

As seen in the table Polymerase I has three activities:

Note that pol I cannot be the main polymerization enzyme because it is way too slow, and shows a low processivity (it falls off the DNA easily making polymerization of large DNA molecules difficult).

DNA pol III functions as a holoenzyme in vivo. (text Figure 25-10a, b, p 984) It is both labile and complex. The subunit composition for the E. coli enzyme is shown in the table below (text Table 25-2, p 983).

E. coli DNA Polymerase III Subunits

Subunit Mass (kD) Gene Function
alpha 130 polC polymerase
epsilon 27.5 dnaQ 3'-exonuclease
theta 8.6 holE alpha, epsilon assembly
tau 71 dnaX holoenzyme assembly on DNA
beta 41 dnaN Sliding clamp-greatly increases processivity.
gamma 47.5 dnaX(Z) gamma-complex
del 39 holA gamma-complex
del' 37 holB  gamma-complex
Chi 17 holC  gamma-complex
psi 15 holD  gamma-complex

Pol III can also be isolated as a core enzyme. In this form it will catalyze polymerization, however its processivity drops from the >5000 bps in the holoenzyme to about 10-15 bps. The holoenzyme also requires ATP to bind to the template/primer.

Pol III differs from Pol I in that it cannot unwind DNA, requiring a series of initiation proteins, unwinding proteins, single stranded binding protein (ssb), etc. as shown in the table below. These work in concert with ATP hydrolysis (2 ATP/bp) to open the DNA double helix in advance of Pol III. Note that the ssb must be stripped off of the DNA strands before Pol III can replicate it.

The actual replication of DNA occurs in the replisome, a complex including two Pol III active sites, one to synthesize the leading strand and one to synthesize the lagging strand. This results in a difficulty since the leading strand is replicated only in the 5'-3' direction, and the complimentary strand is then going the opposite direction. This problem is solved by synthesising a sufficient length of DNA to allow the lagging single strand to loop around and come in parallel, instead of anti-parallel, to the leading strand. Note that after each Okazaki fragment is synthesized by the lagging Pol III, the enzyme must relocate along the lagging strand, by opening and closing the lagging "clamp" to a newly synthesized primer to start the next fragment. (text Figures 25-12, p 984; 25-13, p 987; 25-14, p 988; 25-15, p 989)

E. coli DNA Replication Proteins

Protein Function
DNA polymerase III holoenzyme DNA synthesis

 Replication Initiation Proteins (text Table 25-3, p 986)
DNA gyrase
DNA unwinding (relieves supercoiling induced by replication and DnaB)
ssb
single-stranded binding protein (prevent ssDNA from reannealing behind helicase)
DnaA
Initiation factor (Multimeric complex binds at oriC and causes helix to open [melt] with ATP hydrolysis.)
HU
DNA binding (histone-like). Prevents non-oriC binding of DnaA

Primosome (required to initiate each Okazaki fragment) (text Table 25-4, p 989)
PriA
Primosome assembly, 3'right arrow 5' helicase
PriB
Primosome assembly
PriC
Primosome assembly
DnaB
5'right arrow 3' helicase, unwinds DNA in ATP dependent manner, producing positive supercoiling. Part of prepriming complex.
DnaC
Delivers DnaB to oriC
DnaT
Assists DnaC in delivery of DnaB
Primase (DnaG)
Synthesizes RNA primer
DNA pol I Removes RNA primer, replacing with DNA.
Tus Termination of polymerization at Ter locus opposite oriC

Synthesis of the lagging strand also requires two additional enzymes:


Pathway Diagrams

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Last modified 23 March 2009