Chromosome Chromosome ReplicationReplication

Muh. Nasrum MassiMuh. Nasrum MassiDEPT. MEDICAL MICROBIOLOGY,DEPT. MEDICAL MICROBIOLOGY,

FAC. MEDICINE, HASANUDDIN FAC. MEDICINE, HASANUDDIN UNIVERSITY,UNIVERSITY,MAKASSARMAKASSAR

Chromosome replicates through DNA Chromosome replicates through DNA replication.replication.

DNA replication is a process of copying DNA replication is a process of copying DNA double strand. DNA double strand.

Since DNA are antiparalell and Since DNA are antiparalell and complementary, each strand can serve as complementary, each strand can serve as a template for the new opposite strand.a template for the new opposite strand.

Because a new synthesized DNA double Because a new synthesized DNA double strand consists of an old strand and a new strand consists of an old strand and a new antiparalel double strand, DNA synthesis is antiparalel double strand, DNA synthesis is termed termed semiconservative replicationsemiconservative replication..

Semiconservative ReplicationSemiconservative Replication

DNA replication is made up of three phases:DNA replication is made up of three phases:

1.1. InitiationInitiation, involves recognition of the , involves recognition of the position(s) on a DNA molecule where position(s) on a DNA molecule where replication begins.replication begins.

2.2. ElongationElongation, concerns the events , concerns the events occuring at the replication fork, where the occuring at the replication fork, where the parent nucleotide are copied.parent nucleotide are copied.

3.3. TerminationTermination, occurs when the parent , occurs when the parent molecule has been completely replicated.molecule has been completely replicated.

A. Initiation A. Initiation Initiation of replication is not a random Initiation of replication is not a random

process and always begins at the same process and always begins at the same position(s) on a DNA molecule, these position(s) on a DNA molecule, these points being called the points being called the origins of origins of replicationreplication. .

A circular bacterial genome has a A circular bacterial genome has a singlesingle origin of replication, whilest eukaryotic origin of replication, whilest eukaryotic chromosomes have chromosomes have multiplemultiple origins. origins.

An initiatior protein binds to the origin of An initiatior protein binds to the origin of replication sequence and slightly unwind replication sequence and slightly unwind the double helix using ATP energy. the double helix using ATP energy.

In most cases, DNA replication is In most cases, DNA replication is bidirectional: Two replication forks are bidirectional: Two replication forks are fromed at an origin, and replication fromed at an origin, and replication proceeds simultanously in both direction.proceeds simultanously in both direction.

For circular bacterial DNA this process is For circular bacterial DNA this process is called called theta replicationtheta replication, whilest in linear , whilest in linear eukaryotic DNA this process is called eukaryotic DNA this process is called Y Y replicationreplication..

Theta replicationTheta replication

Y ReplicationY Replication

A.1. Unwinding the DNAA.1. Unwinding the DNA For DNA replication to proceed, the DNA For DNA replication to proceed, the DNA

must unwind to expose the single strands must unwind to expose the single strands to the enzymes responsible for copying to the enzymes responsible for copying them. them.

The proteins responsible for unwinding The proteins responsible for unwinding the DNA are called the DNA are called helicasehelicase. These are . These are enzymes that use the energy of ATP to enzymes that use the energy of ATP to unwind DNA in advance of the replication unwind DNA in advance of the replication fork.fork.

GyraseGyrase is an enzyme that serves as a swivel is an enzyme that serves as a swivel that prevents overwinding of the DNA ahead of that prevents overwinding of the DNA ahead of the replication fork.the replication fork.

Once strand separation begun, molecules of Once strand separation begun, molecules of single strand binding proteinsingle strand binding protein (SSB), quickly (SSB), quickly attach to the exposed single strands at the attach to the exposed single strands at the replication fork, in such a way that they do not replication fork, in such a way that they do not cover the nitrogenous bases.cover the nitrogenous bases.

The SSB molecules hold the separated strands The SSB molecules hold the separated strands in a semiextended position that makes the in a semiextended position that makes the separated DNA strands more accessible to the separated DNA strands more accessible to the DNA replication machinery. DNA replication machinery.

DNA ReplicationDNA Replication

B. ElongationB. Elongation Enzymes capable of adding successive Enzymes capable of adding successive

nucleotides to a growing DNA strand are called nucleotides to a growing DNA strand are called DNA polymeraseDNA polymerase. .

DNA polymerase DNA polymerase requires a templaterequires a template, all use , all use deoxyribonucleoside triphosphatedeoxyribonucleoside triphosphate as their as their substrates.substrates.

Each successive nucleotide is linked to the Each successive nucleotide is linked to the growing chain by a growing chain by a phosphodiester bond phosphodiester bond between the phosphate group on its 5’ carbon and between the phosphate group on its 5’ carbon and the hydroxyl group on the 3’ carbon of the the hydroxyl group on the 3’ carbon of the previous nucleotide. previous nucleotide.

Chain elongation therefore always occurs at Chain elongation therefore always occurs at the 3’ end of a DNA strand, and the strand the 3’ end of a DNA strand, and the strand grow in 5’grow in 5’3’ direction.3’ direction.

Since DNA polymerase is only able to Since DNA polymerase is only able to synthesize DNA in the 5’synthesize DNA in the 5’3’ direction, one 3’ direction, one strand of the double helix can be coupled in strand of the double helix can be coupled in continouscontinous manner ( manner (leading strandleading strand), but ), but replication in the other strand (replication in the other strand (lagging lagging strandstrand) has to be carried out in a ) has to be carried out in a discontinousdiscontinous fashion, as a series of short fashion, as a series of short segments (okazaki fragments) that must be segments (okazaki fragments) that must be ligated together to produce intact daughter ligated together to produce intact daughter strand.strand.

DNA polymerase can not initiate DNA DNA polymerase can not initiate DNA synthesis because it is template dependent. synthesis because it is template dependent.

This means that primers are needed, one to This means that primers are needed, one to initiate complemetary strand synthesis on the initiate complemetary strand synthesis on the leading polynucleotide, and on for every leading polynucleotide, and on for every segment of discontinous DNA synthesized on segment of discontinous DNA synthesized on lagging strand.lagging strand.

The primers are a short piece (3-10 bp) RNAThe primers are a short piece (3-10 bp) RNA Initiation of primers are done by non Initiation of primers are done by non

transcriptional RNA polymerase called transcriptional RNA polymerase called primase. Iprimase. In prokaryotes primase binds with n prokaryotes primase binds with unwinding proteins forming a primosome, but unwinding proteins forming a primosome, but is a part of DNA polymerase is a part of DNA polymerase αα in eukaryotes. in eukaryotes.

In prokaryotes, DNA polymerase involved in In prokaryotes, DNA polymerase involved in replication are DNA polymerase I, II, and III.replication are DNA polymerase I, II, and III.

DNA polymerase IDNA polymerase I consists of one subunit, consists of one subunit, functions in DNA synthesis and DNA repair.functions in DNA synthesis and DNA repair.

DNA polymerase IIDNA polymerase II consists of one consists of one subunit, functions in DNA repair.subunit, functions in DNA repair.

DNA polymerase IIIDNA polymerase III consists of at least 10 consists of at least 10 subunits, and is the subunits, and is the mainmain replicating replicating enzyme. enzyme.

B.1. Prokaryotic DNA B.1. Prokaryotic DNA polymerasepolymerase

B.2. Eukaryotic DNA B.2. Eukaryotic DNA polymerasepolymerase In eukaryotes, DNA polymerase involved in In eukaryotes, DNA polymerase involved in

DNA synthesis are DNA polymerase DNA synthesis are DNA polymerase αα, , ββ, , γγ, , δδ, dan , dan εε

DNA polymerase DNA polymerase αα, consists of 4 , consists of 4 subunits, functions in priming during subunits, functions in priming during replication, contains a primase.replication, contains a primase.

DNA polymerase DNA polymerase ββ, consists of one , consists of one subunit and functions in DNA repair.subunit and functions in DNA repair.

DNA polymerase DNA polymerase γγ, consists of 2 subunits , consists of 2 subunits and functions in mitochondrial DNA and functions in mitochondrial DNA replicationreplication

DNA polymerase DNA polymerase δδ, consists of 2 to three , consists of 2 to three subunits, functions as subunits, functions as mainmain replicating replicating enzyme. enzyme.

DNA polymerase DNA polymerase εε, consists of at least , consists of at least one subunit, function in DNA replication, one subunit, function in DNA replication, but precise function is still unknown. but precise function is still unknown.

C. TerminationC. Termination Termination occurs when DNA replication Termination occurs when DNA replication

forks meet one another or run to the end forks meet one another or run to the end of a linear DNA molecule. of a linear DNA molecule.

Also, termination may occur when a Also, termination may occur when a replication fork is deliberately stopped by replication fork is deliberately stopped by a special protein, called a a special protein, called a replication replication terminator proteinterminator protein, that binds to specific , that binds to specific sites on a DNA molecule sites on a DNA molecule

When the polymerase reaches the end of a linear When the polymerase reaches the end of a linear DNA molecule, there is a potential problem due DNA molecule, there is a potential problem due to the antiparallel structure of DNA.to the antiparallel structure of DNA.

Because an RNA primer must be regularly laid Because an RNA primer must be regularly laid down on the lagging strand, the last section of down on the lagging strand, the last section of the lagging-strand DNA cannot be replicated the lagging-strand DNA cannot be replicated because there is no DNA template for the primer because there is no DNA template for the primer to be synthesized on. to be synthesized on.

To solve this problem, the ends of most To solve this problem, the ends of most chromosomes consist of chromosomes consist of noncodingnoncoding DNA DNA that that contains repeat sequences. contains repeat sequences.

The end of a linear chromosome is called the The end of a linear chromosome is called the telomeretelomere. .

Cells can endure the shortening of the Cells can endure the shortening of the chromosome at the telomere to a degree, since chromosome at the telomere to a degree, since it's necessary for chromosome stability. it's necessary for chromosome stability.

Many cells use an enzyme called Many cells use an enzyme called telomerasetelomerase that adds the repeat units to the end of the that adds the repeat units to the end of the chromosome so the ends do not become too chromosome so the ends do not become too short after multiple rounds of DNA replication.short after multiple rounds of DNA replication.

Many simple, single-celled organisms overcome Many simple, single-celled organisms overcome the whole problem by having circular the whole problem by having circular chromosomes.chromosomes.

Chromosome Chromosome Organization and Organization and EvolutionEvolution

Muh. Nasrum MassiMuh. Nasrum MassiDEPT. MEDICAL MICROBIOLOGY,DEPT. MEDICAL MICROBIOLOGY,

FAC. MEDICINE, HASANUDDIN UNIVERSITY,FAC. MEDICINE, HASANUDDIN UNIVERSITY,MAKASSARMAKASSAR

A. Chromosom A. Chromosom OrganizationOrganization

Beside genes containing codes for Beside genes containing codes for protein synthesis (protein synthesis (exonsexons), DNA also ), DNA also contain non-coding proteins like contain non-coding proteins like intronsintrons, , promoterspromoters, and , and enhancersenhancers..

A.1. ExonsA.1. Exons An An exonexon is any region of is any region of DNADNA within a within a

gene that is transcribed to the final gene that is transcribed to the final messenger RNAmessenger RNA (mRNA) molecule, (mRNA) molecule, rather than being rather than being splicedspliced out from the out from the transcribedtranscribed RNA molecule. Exons of RNA molecule. Exons of many eukaryotic genes interleave with many eukaryotic genes interleave with segments of non-coding DNA (segments of non-coding DNA (intronsintrons). ).

A.2. IntronsA.2. Introns IntronsIntrons are sections of are sections of DNADNA that will be that will be

splicedspliced out after out after transcriptiontranscription, but before , but before the the RNARNA is used. Introns are common in is used. Introns are common in eukaryoticeukaryotic RNAs of all types, but are RNAs of all types, but are found in found in prokaryoticprokaryotic tRNAtRNA and and rRNArRNA genes only. genes only.

The number and length of introns varies The number and length of introns varies widely among widely among speciesspecies and among genes and among genes within the same species. within the same species.

A.3. PromoterA.3. Promoter a a promoterpromoter is a is a DNADNA sequence that sequence that

enables a enables a genegene to be transcribed. The to be transcribed. The promoter is recognized by RNA promoter is recognized by RNA polymerase, which then initiates polymerase, which then initiates transcription. In RNA synthesis, transcription. In RNA synthesis, promoters are a means to demarcate promoters are a means to demarcate which genes should be used for which genes should be used for messenger RNA creation - and, by messenger RNA creation - and, by extension, control which proteins the cell extension, control which proteins the cell manufactures. manufactures.

A.4. EnhancerA.4. Enhancer an an enhancerenhancer is a short region of DNA is a short region of DNA

that can be bound with proteins (namely, that can be bound with proteins (namely, the trans-acting factors, much like a set the trans-acting factors, much like a set of transcription factors) to enhance of transcription factors) to enhance transcription levels of genes (hence the transcription levels of genes (hence the name) in a gene-cluster. An enhancer name) in a gene-cluster. An enhancer does not need to be particularly close to does not need to be particularly close to the genes it acts on, and need not be the genes it acts on, and need not be located on the same chromosome located on the same chromosome

An enhancer does not need to bind close to An enhancer does not need to bind close to the transcription initiation site to affect its the transcription initiation site to affect its transcription, as some have been found to transcription, as some have been found to bind several hundred thousand base pairs bind several hundred thousand base pairs upstream or downstream of the start site.upstream or downstream of the start site.

Enhancers do not act on the promoter Enhancers do not act on the promoter region itself, but bind to activator proteins. region itself, but bind to activator proteins. These activator proteins interact with the These activator proteins interact with the mediator. The mediator is the protein that mediator. The mediator is the protein that communicates with the polymerase II and communicates with the polymerase II and the general transcription factors. the general transcription factors.

Enhancers can also be found within introns. Enhancers can also be found within introns.

B. Chromosome EvolutionB. Chromosome Evolution Genomes are dynamic entities that Genomes are dynamic entities that

evolve over time due to the cumulative evolve over time due to the cumulative effects of small scale sequence effects of small scale sequence alterations caused by alterations caused by mutationmutation, and , and larger scale rearrangements arising from larger scale rearrangements arising from recombination.recombination.

B.1. MutationB.1. Mutation A mutation is a change in the nucleotide A mutation is a change in the nucleotide

sequence of a short region of a genome. sequence of a short region of a genome. Many mutations are Many mutations are point mutationspoint mutations that that

replace one nucleotide with another.replace one nucleotide with another. Other mutation involve Other mutation involve insertioninsertion or or deletiondeletion of of

one or a few nucleotides.one or a few nucleotides. Mutations result either from errors in DNA Mutations result either from errors in DNA

replication or from the damaging effects of replication or from the damaging effects of mutagensmutagens such as chemicals and radiation such as chemicals and radiation that react with DNA and change the structures that react with DNA and change the structures of individual nucleotides.of individual nucleotides.

A A mutagenmutagen is a chemical or physical agent is a chemical or physical agent that cause mutation.that cause mutation.

Chemical mutagens are base analogs, Chemical mutagens are base analogs, deaminating agents, alkylating agents, and deaminating agents, alkylating agents, and intercalating agents.intercalating agents.

Physical mutagens are UV radiation, Physical mutagens are UV radiation, ionizing radiation, and heat.ionizing radiation, and heat.

The effects of mutation to the genome are The effects of mutation to the genome are silent mutation, missense mutation, and non silent mutation, missense mutation, and non sense mutation. sense mutation.

Silent mutationSilent mutation is the mutation where the is the mutation where the new codon specifies the same amino acid new codon specifies the same amino acid as the unmutated codon.as the unmutated codon.

Missense mutationMissense mutation, where mutation , where mutation altering the codon so that it specifies a altering the codon so that it specifies a different amino acids.different amino acids.

Non sense mutationNon sense mutation results in a shortened results in a shortened protein because translation of the mRNA protein because translation of the mRNA stops at a new termination codon rather stops at a new termination codon rather then proceeding to the correct termination then proceeding to the correct termination codon which is further downstream.codon which is further downstream.

All cells possess All cells possess DNA repair enzymesDNA repair enzymes that attempt to minimize the number of that attempt to minimize the number of mutations that occur. mutations that occur.

Most cells possess five different categories Most cells possess five different categories of DNA repair system: direct repair system, of DNA repair system: direct repair system, base excision repair, nucleotide excision base excision repair, nucleotide excision repair, mismatch repair, and recombination repair, mismatch repair, and recombination repair.repair.

Direct repair systemDirect repair system, act directly on , act directly on damaged nucleotides, converting each one damaged nucleotides, converting each one back to its original structure. back to its original structure.

Base excisionBase excision repair, involves removal of repair, involves removal of a damaged nucleotide base, excision of a a damaged nucleotide base, excision of a short peace of the polynucleotide around short peace of the polynucleotide around the AP site thus created, and resynthesis the AP site thus created, and resynthesis with a DNA polymerase.with a DNA polymerase.

Nucleotide excision repairNucleotide excision repair, is similar to , is similar to base excision repair but is not preceeded base excision repair but is not preceeded by removal of a damaged base and can act by removal of a damaged base and can act on more substantially damaged areas of on more substantially damaged areas of DNA.DNA.

Mismatch repairMismatch repair, corrects errors of , corrects errors of replication, again by excising a stretch of replication, again by excising a stretch of single-stranded DNA containing the single-stranded DNA containing the offending nucleotide and then repairing the offending nucleotide and then repairing the resulting gap.resulting gap.

Recombination repairRecombination repair, is used to mend , is used to mend doublestrand breaks. doublestrand breaks.

B.2. RecombinationB.2. Recombination Recombination results in a restructuring Recombination results in a restructuring

of part of a genome, for example by of part of a genome, for example by exchange of segments of homologous exchange of segments of homologous chromosomes during meiosis or by chromosomes during meiosis or by transposition of a mobile element from transposition of a mobile element from one position to another within a one position to another within a chromosome or between chromosomes. chromosome or between chromosomes.