Molecular BiologyReplication: Process & Machinery

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190018. India.

• Proposed by Francis Crick in 1950s to suggest that there was “the unidirectional flow of genetic information”

• Starts from the DNA and ends in proteins via RNA

• It included three main process:

• Replication: For new DNA synthesis from template DNA

• Transcription: For synthesis of RNA on the information of DNA

• Translation: Synthesis of Protein using information from RNA

Central Dogma

• Subjected to change by incorporating the processes of reversetranscription- which involves synthesis of DNA from RNA (inretroviruses), RNA replication & editing- involves RNAsynthesis or editing form RNA & Prions- involves proteinguided misfolding of proteins

Central Dogma

GENE EXPRESSION• Prokaryotes

DNA

• Replication

RNA

• Transcription

Proteins

• Translation

GENE EXPRESSION• Eukaryotes

DNA

• Replication

Pre RNA

• Transcription

RNA

• Post Transcriptional Editing

• Post Transcriptional Modifications

Pre Proteins

• Translation

Proteins

• Post Translation Modifications

• Pre Folding Modifications

REPLICATION• THE PROCESS

• Replication can be simply defined as the duplication of the genome

• The genome which is duplicated is called as the parental genome and the copies are called as the daughter genomes

• The process of replication in itself is very complex involving a number of enzymes - the central of which are polymerases

• During the replication process, most of the enzymes involved directly or indirectly are present in the form of dynamic complex called as the replisome

REPLICATION• THE PROCESS

• Replication process takes place in 5’→3’ direction; with 3’-OH end of the daughter strand acting as the extension end to which new DNTPs are added

• The strand which serves as template is read from 3’-end towards its 5’- end

REPLICATION• THE PROCESS

• The strand in which 5’→3’ DNA synthesis proceeds in same direction as that of the replication fork is called as Leading Strand

• The strand in which 5’→3’ DNA synthesis proceeds in opposite direction as that of the replication fork is called as Lagging Strand

REPLICATION• THE PROCESS

• Leading Strand directs the synthesis of new daughter DNA in continuous manner

• Lagging Strand directs the synthesis of new daughter DNA in discontinuous manner • The fragmental synthesis of DNA over lagging strand makes the

whole DNA synthesis as semi-discontinous

• The fragments are referred to as “Okazaki fragments”(ReigiOkazaki; 1960s)

REPLICATION• THE PROCESS

• DNA synthesis is actually a nucleophilic attack of the 3’-hydroxyl group on the incoming DNTPs

• The nucleophile is the 3’-hydroxyl group of the nucleotide at the 3’- end of the growing strand

• Nucleophilic attack occurs at the α phosphorus of the incoming deoxynucleoside 5’- triphosphate

REPLICATION• THE PROCESSIVITY

• Rate at which new nucleotides are being added to the growing daughter strand

• The average number of nucleotides added by the DNA polymerase before it is disassociated from the template

• Nucleophilic attack vary greatly in processivity:

• from few ~ many thousands

REPLICATION• THE ACCURACY

• Overall fidelity of replication is given by its error rate; which is once for every 109-1010

• E coli : 4.6x106 – it means error rate of only once per 1000 to 10000 replications

• The overall geometry of the complimentary bases i.e., A=T and G=C; which is checked by polymerase before moving to next adjacent site

• The presence of 3’→5’ exonuclease activity; which double checks each nucleotide after it is added against the template strand in new strand. (Proofreading). It increases the accuracy by 102 to 103 fold

• Base pair interactions with polymerases

Replication: The Machinery

REPLICATION• THE MACHINERY

• Overall enzymes and proteins needed for synthesis of new DNA are referred to as Replisome

• Comprises of DNA Polymerase together with accessory proteins and enzymes

The main components of the replisome

Component FunctionDNA polymerases

DNA helicaseDNA ligaseDNA primaseDNA topoisomerase

RnaseHSSBP’s

Synthesizes DNA, repairs gaps in lagging strand. Has many forms eachperforming different unique functionUnwinds DNA ahead of replication forkJoins the DNA fragments during lagging strand synthesisSynthesis primer needed to initiate the DNA synthesis by polymeraseChanges the linking number of DNA and hence relaxes the torsional straincaused by helicase activityRemoves RNA primers from the lagging strandKeeps the single strands in linear form

The PolymerasesIrrespective of the source all of DNA polymerases possess some basis properties:

• All catalyze the synthesis of new DNA in 5׳3→׳ direction, with the free 3’ OH group being the point at which the DNA is elongated. Thus all polymerases possess a 5׳3→׳ polymerase activity

• All are template dependent without which they cannot synthesize the new DNA strand. Thus they need to be told when, where & what nucleotide to incorporate for the synthesis of new DNA strand

• All require a primer which provides the free 3׳ OH group to which nucleotide can be added. The free end of primer is called as the primer terminus

• Usually all major polymerases possess 3׳ 5→׳exonucleaseactivity; which is required to check for the correct nucleotide inserted during DNA synthesis - the process called as proofreading

E Coli Polymerases• E. coli possess genes coding for five different polymerases but

three are mainly common - I, II & III

• DNA polymerase III is the principle polymerase and unlike others is a multimeric complex of 18 subinunits{α2ε2θ2τ2β4γ2δδ́χψ} of ten types

• The holoenzyme called as DNA polymerase III* is subassembly of 14 subunits consisting of

• two core polymerase of 8 subunits α2ε2θ2τ2 (each core polymerase is αεθ while τ acts as dimerisation subunit )and

• α has polymerase activity

• ε has proof reading activity

• clamp-loading complex of six subunits γ2δδχ́ψ

• DNA polymerase III* has very low processivity but by binding of dimer of β subunit to each core polymerase, processivity is increased to greater than 500000

E Coli Polymerases

E Coli PolymerasesComparison of DNA polymerases of E. coli

Pol I Pol II Pol III

Gene

Structure

Polymerization rate

Processivity

׳3→׳5 polymerase׳5→׳3 exonuclease׳3→׳5 exonuclease

polA

Polypeptide

16-20

3-200

YesYesYes

polB

Polypeptide

40

1500

YesYesNo

polC, dnaE, dnaN, dnaQ etc

Multimeric complex

250-1000

>500000

YesYesNo

E Coli Polymerases• DNA polymerase I is unique

polymerase because it possesses an additional 5’→3’ exonucleaseactivity in its N-terminal domain

• This could be separated from the main enzyme by mild protease treatment without affecting its 5’→3’ polymerase & 3’→5’ exonuclease activity located within the large fragment called as Klenow fragment (68kD)

• This polymerase plays role in nick translation

Questions?