Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

replication

Overview of DNA SynthesisOverview of DNA Synthesis

DNA polymerases synthesize DNA polymerases synthesize new strands in 5new strands in 5’’ to 3 to 3’’ direction.direction.

Primase makes RNA primer.Primase makes RNA primer.

Lagging strand DNA consists Lagging strand DNA consists of Okazaki fragments.of Okazaki fragments.

In In E. coliE. coli, pol I fills in gaps in , pol I fills in gaps in the lagging strand and the lagging strand and removes RNA primer.removes RNA primer.

Fragments are joined by DNA Fragments are joined by DNA ligase.ligase.

DNA Replication at Growing ForkDNA Replication at Growing Fork

DNA polymerases add DNA polymerases add nucleotides in nucleotides in 55’’ to 3 to 3’’ direction direction. .

Because of antiparallel nature, Because of antiparallel nature, synthesis of DNA is synthesis of DNA is continuous continuous for one strand and for one strand and discontinuous discontinuous for the other for the other strand.strand.

DNA Replication:DNA Replication:Synthesis of Synthesis of Lagging StrandLagging Strand

Several components Several components and steps are and steps are involved in the involved in the discontinuous discontinuous synthesis of the synthesis of the lagging strand. lagging strand.

Note that DNA Note that DNA polymerases move in polymerases move in 33’’ to 5 to 5’’ direction direction on on the template DNA the template DNA sequence. sequence.

DNA Replication:DNA Replication:Synthesis of Synthesis of Lagging StrandLagging Strand

DNA extended from DNA extended from primers are called primers are called OkazakiOkazaki fragmentsfragments. .

In In E. coliE. coli, pol I , pol I removes RNA primers removes RNA primers and fills in the gaps and fills in the gaps left in lagging left in lagging strands.strands.

DNA ligaseDNA ligase joins joins these pieces.these pieces.

Replisome and Accessory Replisome and Accessory ProteinsProteins

pol III holoenzyme is a pol III holoenzyme is a complex of many complex of many different proteins.different proteins.

Looping of Looping of template DNA template DNA for the lagging for the lagging strand allows strand allows the two new the two new strands to be strands to be synthesized synthesized by one dimer.by one dimer.

Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths etet alal., 2012. ., 2012.

Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths etet alal., 2012. ., 2012.

Priming DNA SynthesisPriming DNA Synthesis

DNA polymerases can DNA polymerases can extendextend (but cannot start) (but cannot start) a chain.a chain.

DNA DNA polymerase polymerase extends RNA extends RNA primerprimer with with DNA.DNA.

PrimasePrimase enzyme enzyme makes short makes short RNA RNA primerprimer sequence sequence complementary to complementary to template DNA.template DNA.

PrimosomePrimosome is a is a set of proteins set of proteins that are that are involved in the involved in the synthesis of synthesis of RNA primers. RNA primers.

Supercoiling results from separation of template Supercoiling results from separation of template strands during DNA replication.strands during DNA replication.

Helicase enzymes disrupt Helicase enzymes disrupt hydrogen bonding hydrogen bonding between complementary between complementary bases.bases.

Single-stranded binding Single-stranded binding protein stabilizes protein stabilizes unwound DNA.unwound DNA.

Unwound condition Unwound condition increases twisting and increases twisting and coiling, which can be coiling, which can be relaxed by relaxed by topoisomerases (such as topoisomerases (such as DNA gyrase).DNA gyrase).

Topoisomerases can either Topoisomerases can either create or relax supercoiling.create or relax supercoiling.

They can also induce or They can also induce or remove knots.remove knots.

Helicases and Helicases and TopoisomerasesTopoisomerases

Chromatin assembly factor I (CAF-I) and Chromatin assembly factor I (CAF-I) and histones are delivered to the replication histones are delivered to the replication fork.fork.

CAF-I and histones bind to proliferating cell CAF-I and histones bind to proliferating cell nuclear antigen (PCNA), the eukaryotic nuclear antigen (PCNA), the eukaryotic version of clamp protein.version of clamp protein.

Nucleosome assembly follows thereafter.Nucleosome assembly follows thereafter.

Refer to Figure 7-23 from Introduction to Genetic Analysis, Griffiths Refer to Figure 7-23 from Introduction to Genetic Analysis, Griffiths etet alal., 2012. ., 2012.

Overview of DNA SynthesisOverview of DNA Synthesis

DNA polymerases synthesize DNA polymerases synthesize new strands in 5new strands in 5’’ to 3 to 3’’ direction.direction.

Primase makes RNA primer.Primase makes RNA primer.

Lagging strand DNA consists Lagging strand DNA consists of Okazaki fragments.of Okazaki fragments.

In In E. coliE. coli, pol I fills in gaps in , pol I fills in gaps in the lagging strand and the lagging strand and removes RNA primer.removes RNA primer.

Fragments are joined by DNA Fragments are joined by DNA ligase.ligase.

Prokaryotes:Prokaryotes:

Fixed originFixed originDnaA proteinsDnaA proteinsDnaB (helicase)DnaB (helicase)

Eukaryotes:Eukaryotes:

Multiple originsMultiple originsORC protein complexORC protein complexCdc6 and Cdt1Cdc6 and Cdt1MCM complex MCM complex (helicase)(helicase)

Initiation at Origin of ReplicationInitiation at Origin of Replication