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Nucleases

Enzymes which cleave nucleic acids (RNA and DNA)are called nucleases.

Enzymes that cleave DNA are called DNases; thosethat cleave RNA are called RNases.

Act on single stranded or double stranded substrates.Exonucleases cleave from one end.Endonucleases cleave internal sites.Exonucleases could be specific for 5 to 3 or 3 to 5direction.

Sequence specific or unspecific.

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DNA Polymerases

At least 5 DNA polymerases: I, II, III, IV and V in E. coli.Pol I, the most abundant (~90% of the Pol activity). Addsnucleotides at the rate of 16-20 nucleotides per second.

The E. coli genome (4.6 X 106): copied in

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Klenow fragment of DNA pol I: Lacks the 3 to 5 exonuclease domain

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Priming

DNA replication is primed by a short stretch of RNA that issynthesized by primase, an RNA polymerase.

The RNA primer is removed at a later stage of replication.

Some double stranded phage genomes, linear plasmids etc. overcome theproblem of replicating the ends of linear DNA by protein priming.

Terminal protein (TP) is covalently attached to the 5 end. Ser/Thr/Tyr residuefrom TP provides the OH group for DNA polymerase.

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DNA polymerase mechanism

Two metal ions (typically, Mg++) participate in the reaction.

One metal ion coordinates with the 3' OH of the primer; and theother with the dNTP.

The hydroxyl group of the primer attacks the phosphate group toform a new O-P bond.

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Shape selectivity

The binding of a dNTP to DNA polymerase induces aconformational change, generating a tight pocket.

Conformational change occurs when the dNTP corresponds tothe Watson-Crick partner of the template base.

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Figure 5-4 Molecular Biology of the Cell( Garland Science 2008)

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Fidelity of DNA Replication

Replication proceeds with an extraordinary degreeof fidelity.

In E. coli, a mistake is made only once in 109 to1010nucleotides added.

For E. coli (chromosome size- 4.6 x 106 bp), thismeans that an error occurs only once in 1,000 to10,000 replications.

Hydrogen bonding schemeGeometry of the correct base pair3 to 5 exonuclease activity (proofreading)DNA mismatch repair systems

104to 105

102to 103

102to 103

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The standard A :T and G:C basepairs have very similar

geometries which fit the activesite.

The geometry of incorrectlypaired bases can exclude themfrom the active site.

Contribution of base-pair

geometry to the fidelity of

replication

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!"#$%&'( *&++",- ./&+'"0 12 *+$0/&3- 4$56/"3$07+8 9+(:($;5%- - ??@9=

Mispairing due to tautomerization

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!"#$%&'( !$*+ ,-'./"0-*12&+ 3.$4"0-. 52"**+ 671/84-('9 7::;+ ?@

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Figure 5-9 Molecular Biology of the Cell( Garland Science 2008)

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Okazaki fragments

At a replication fork both strands are synthesized in the 5' - 3' direction.The leading strand is synthesized continuously whereas the lagging strand issynthesized in short pieces termed Okazaki fragments (1000-2000 nucleotideslong in prokaryotes; 150-200 nucleotides long in eukaryotes).

AB

;B

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DNA ligase catalyzes the joining of one DNAstrand with a free 3' -hydroxyl group to anotherwith a free 5' -phosphate group.

In eukaryotes and archaea, ATP is cleaved toAMP & PPi.

In bacteria, NAD+ is cleaved to AMP and NMN.Ligase seals breaks in double-stranded DNAmolecules.

DNA Ligase: Joins the ends of DNA in the duplex

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Replication fork

A schematic view of the arrangement of DNA polymerase IIIand associated proteins present in replicating DNA.

The helicase separates the two strands of the parent doublehelix. allowing DNA polymerases to use each strand as a

template for DNA synthesis.

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DNA polymerase holoenzyme

Each holoenzyme consists of two copies of the polymerasecore enzyme (!, !, and "subunits) and two copies of the "subunit, linked to a central structure.

The central structure includes the clamp-loader complex andthe hexameric helicase DnaB

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Trombone model

The replication of the leading and lagging strands is coordinatedby the looping out of the lagging strand.DNA replicase system or Replisome.

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Structure of a sliding DNA clamp

The dimeric "subunit of DNA polymerase III forms a ringthat surrounds the DNA duplex.There is a central cavity through which the DNA templateslides.

The polymerase enzyme is able to move without falling offthe DNA substrate.

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Origin of Chromosome replication, oriC

A 245 bp region which is highly conserved amongbacterial replication origins.

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C!D3 E9F-94-9G 8H"0"91I

Initiation

Loading of the DnaB helicase: key step in replicationinitiation.

DnaB migrates along the single-stranded DNA in the 5-3'direction, and unwinds the DNA as it travels (~5000 rpm).DNA gyrase (DNA topoisomerase II) relieves the topologicalstress induced ahead of the fork.

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Elongation

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The terminus region contains multiple copies of a Ter sequence, which bindsTus(terminus utilization substance) protein.

When either replication fork encounters a functional Tus-Ter complex, it halts;the other fork halts when it meets the first (arrested) fork.

The final few hundred base pairs of DNA between these large proteincomplexes are then replicated by an as yet unknown mechanism).

Topo IV (a type II topoisomerase) separates the catenated circles in E. coli.

Termination

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Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Dideoxynucleotide

no hydroxyl group at 3 end

prevents strand extension

.AD

9D

4EF:

3

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Chain Terminator Basics

G&+#"7

:H7"%(

443

44J

44K

44L

I&J"'"( G"+3$%&75+0443

E 44K

E. 44J

E.* 44L

G*.E

dN : ddN100 : 1

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KLE F"MN"%6$%#

O2 F7&+7 &7 ?+$3"+ P+"07+$6;5%0$7"Q

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/,?XYYRRR285N7NJ"2653YR&76/Z[\W37I]?#^AI=

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Replication in eukaryotes

Human system requires~30,000 origins of replications.

In humans, 23 pairs of linearchromosomes