dna replication, repair and recombination

DNA REPLICATION, REPAIR AND RECOMBINATION

Nanjing Foreign Language School International Centre Tutoring Club — Biology

EliteMike Chen

Basic Genetic Mechanisms:

Based on Molecular Biology of the Cell 6th edition

OBJECTIVE

• How does DNA replicates itself?• What can go wrong if DNA sequence is changed?• How to ensure we can have identical copies?• How to deal with replication mistakes?• Why DNA replication mechanism has served rules?• How to have high fidelity of DNA replication?• What are the possible damages can DNA suffer and how to repair them?• How to recombine broken chromosomes?• How many options do we have to repair DNA and which one in more favourable?• How do know to use which way to repair?• How cell uses mechanism to perform other genetic change besides repair?

After this lesson, you should hopefully know…….

THE MAINTENANCE OF DNA SEQUENCEMutation: permanent change in the DNA sequence.

Mutation rate is one nucleotide change per 108 nucleotides per human generation. (70 nucleotides of each offspring)Mutation rate is extremely low.

http://evolution.berkeley.edu/evolibrary/article/evo_20

http://evolution.berkeley.edu/evolibrary/article/evo_20

THE MAINTENANCE OF DNA SEQUENCEGerm cells: transmit genetic information from parent to offspringSomatic cells:transit genetic information form the body of the organismChange in somatic cells may lead to cancer

Molecular biology of the cell 6/e ©Garland Science(2015)

DNA REPLICATION MECHANISMS

Semi-conservative replication

PROTEINSDNA REPLICATION MECHANISMS

DNA polymeraseDNA primaseDNA helicase

Single-strand DNA-binding (SSB) proteinsDNA ligase

Polymerase clampclamp loader

strand-directed mismatch repair systemDNA topoisomerase IDNA topoisomerase II

DNA REPLICATION MECHANISMS• DNA polymerase catalyses the stepwise addition of a

deoxynucleotide to the 3’ -OH end of the polynucleotide chain• The free nucleotide served as substrates for this enzyme were found

to be deoxynucleotide triphosphate• The reaction is driven by a large favourable free-energy change,

caused by the release of pyrophosphate• The newly synthesised DNA strand therefore polymerised in the 5’-to-

3’ direction only


DNA REPLICATION MECHANISMSProofreading ability of DNA polymerase

• The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favourable.

• The enzyme must undergo a conformational change in which its “grip” tightens around the active site. It makes the nucleotide to double check the base-paired geometry

• DNA molecules with a mismatched (improperly base-paired) nucleotide at the 3ʹ-OH end of the primer strand are not effective as templates because the polymerase has difficulty extending such a strand.

• DNA polymerase in this case will turn into editing mode, turning into 3’-to-5’ proofreading exonuclease clipping off any unpaired or misfired nucleotides.

• DNA polymerase functions as a “self-correcting” enzyme that removes its own polymerisation errors as it moves along the DNA

DNA REPLICATION MECHANISMSIf there were a DNA polymerase that added deoxyribonucleotide triphosphates in the 3ʹ-to-5ʹ direction, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerisation could not be simply hydrolysed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis.

http://www.sparknotes.com/biology/molecular/dnareplicationandrepair/section3.rhtml

http://www.sparknotes.com/biology/molecular/dnareplicationandrepair/section3.rhtml


Fidelity of DNA replication


DNA REPLICATION MECHANISMSLeading and Lagging Strand

RNA primer• DNA primases adds a RNA primer on the lagging strand for about 10

nucleotides long at the intervals of 100-200 nucleotides• Any enzyme that starts a new chain cannot be self-correct. So reason of

using a RNA primer instead of a DNA primer is to keep the accuracy of replication. DNA primer may make mistakes without self-correcting mechanism. RNA primer will be efficiently removed ad replaced


DNA helicases were first isolated as proteins that hydrolysed ATP when they are bound to single strand of DNA. The hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work.

DNA REPLICATION MECHANISMSSingle-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilising the unwound, single-strand conformation.

DNA REPLICATION MECHANISMSOn their own, most DNA polymerase will synthesis only a short distance before falling off. This property allows DNA polymerase to be recycled so quickly on the lagging strandA sliding clamp helps the DNA polymerase sticks firmly on the DNA while moving. The clamp is a type of accessory protein (PCNA for eukayotes)The clamp has a large ring shape around the DNA double helix. The assembly of the clamp requires ATP hydrolysis by a special protein, called clamp loader, which hydrolyses ATP as it loads the clamp on to a primer-templates junction.


At the front of the replication fork, DNA helicase opens the DNA helix. Two DNA polymerase molecules work at the fork, one on the leading strand and one on the lagging strand. Whereas the DNA polymerase molecule on the leading strand can operate in a continuous fashion, the DNA polymerase molecule on the lagging strand must restart at short intervals, using a short RNA primer made by a DNA primase molecule. The close association of all these protein components increases the efficiency of replication and is made possible by a folding back of the lagging strand. This arrangement also facilitates the loading of the polymerase clamp each time that an Okazaki fragment is synthesised: the clamp loader and the lagging-strand DNA polymerase molecule are kept in place as a part of the protein machine even when they detach from their DNA template.

DNA REPLICATION MECHANISMSStrand-directed mismatch repair system detects the potential for distortion in the DNA helix from the misfit between non complementary base pairs.

Having a defective copy of mismatch repair genes in human can be very dangerous, it may lead to hereditary nonpolyposis colon cancer (HNPCC) due to rapid accumulation of mutations.


DNA REPLICATION MECHANISMSIn E.coli, methylation of all A residues in the sequence GATC is used to distinguish between the old strand and the newly made strand.In eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks and such nicks (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand. Speed of DNA ligase is smaller than mismatch repair system

Jeungphill Hanne, Jiaquan Liu, Jong-Bong Lee, Richard Fishel, Single-molecule FRET Studies on DNA Mismatch Repair, International Journal of Biophysics , Vol. 3 No. 1A, 2013, pp. 18-38. doi: 10.5923/s.biophysics.201311.03.

DNA REPLICATION MECHANISMSAs replication fork moves along the DNA double helix, it creates a winding problem. For every 10 pairs of nucleotides being replication, one turn of the DNA helix must be completed. However, turing the helix is energetically unfavourable, which will create overwound of DNA in front of replication fork.

DNA topoisomerase is used to solve this problem. It can be viewed as a reversible nuclease that adds itself covalently to DNA backbone, breaking phosphdiester bond.


DNA REPLICATION MECHANISMSTopoisomerase I produces a transient single-strand break, which allows two sections of DNA rotates freely to each other.

DNA REPLICATION MECHANISMSTopoisomerase II produces a transient double-strand break, it breaks one double helix reversibly to create a gate, inducing a nearby DNA helix to pass through the gate and then closes it.

DNA REPLICATION IN CHROMOSOMESDNA replication begins at replication origin.DNA sequences that can serve as an origin of replication are found to contain: binding site for a large, multisubunit initiator protein called ORC (origin recognition complex), a stretch of DNA that is rich in As and Ts, at least one binding site for protein that facilitate ORC.

DNA REPLICATION IN CHROMOSOMESIn brief, during G1 phase, the replicative helicases are loaded onto DNA next to ORC to create a prereplicative complex. Then, upon passage from G1 phase to S phase, specialized protein kinases come into play to activate the helicases. The resulting opening of the double helix allows the loading of the remaining replication proteins, including the DNA polymerases. The protein kinases that trigger DNA replication simultaneously prevent assembly of new prereplicative complexes until the next M phase resets the entire cycle This strategy provides a single window of opportunity for prereplicative complexes to form; thus ensure all DNA is copied once only.


DNA REPLICATION IN CHROMOSOMESBacteria only has a single origin of DNA replication

In human, replication of average-sized chromosome will take 35 days if there is only a single replication origin. In eukaryotes, there are multiple replication origin.

DNA REPLICATION IN CHROMOSOMESHistone proteins are required to package DNA and they are usually made only in the S phage during DNA replication.When a nucleosome is traversed by a replication fork, the histone octamer appears to be broken into an H3-H4 tetramer and two H2A-H2B dimers The H3-H4 tetramer remains loosely associated with DNA and is distributed at random to one or the other daughter duplex, but the H2A-H2B dimers are released completely from DNA.

Clément, Camille, and Geneviève Almouzni. "MCM2 Binding to Histones H3–H4 and ASF1 Supports a Tetramer-to-dimer Model for

Histone Inheritance at the Replication Fork." Nat Struct Mol Biol Nature Structural & Molecular Biology 22.8 (2015): 587-89. Web.

DNA REPLICATION IN CHROMOSOMESThe orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers behind a replication fork requires histone chaperonesThe histone chaperones, along with their cargoes (histone proteins), are directed to newly replicated DNA through a specific interaction with the eukaryotic sliding clamp called PCNA


DNA REPLICATION IN CHROMOSOMES

As DNA polymerase δ discontinuously synthesizes the lagging strand, the length of each Okazaki fragment is determined by the point at which DNA polymerase δ is blocked by a newly formed nucleosome. This explains why the length of Okazaki fragments in eukaryotes (~200 nucleotides) is approximately the same as the nucleosome repeat length.

Kurth, Isabel, and Mike O’Donnell. "New Insights into Replisome Fluidity during Chromosome Replication." Trends in Biochemical Sciences 38.4 (2013): 195-203. Web.

DNA REPLICATION IN CHROMOSOMESWhen the replication fork reaches an end of a linear chromosome, the final RNA primer synthesised on the lagging strand cannot be replaced by DNA because there is no 3’-OH end available for repair polymerase, which means DNA will lost a part of its end during every DNA replicationIn bacteria, circular DNA solves this problem. In eukaryotes, specialised nucleotide sequence at the end of the chromosomes called telomeres can solved this problem. Telomeres contain many tandem repeat, in human is GGGTTA

DNA REPLICATION IN CHROMOSOMESTelomere DNA sequences are recognised by sequence-specific DNA-binding proteins that attract an enzyme, called telomerase Telomerase recognises the tip of an existing telomere DNA repeat sequence and elongates it in the 5ʹ-to-3ʹ direction, using an RNA template that is a component of the enzyme itself to synthesise new copies of the repeat

DNA REPLICATION IN CHROMOSOMESTelomeres must clearly be distinguished from these accidental breaks; otherwise the cell will attempt to “repair” telomeres, causing chromosome fusions and other genetic abnormalities. A specialised nuclease chews back the 5ʹ end of a telomere leaving a protruding single-strand end. This protruding end—in combination with the GGGTTA repeats in telomeres—attracts a group of proteins that form a protective chromosome cap known as shelterin.


DNA REPAIRDNA double helix can be damaged in many ways, including deprivation (loss of guanine/adenine), deamination (cytosine to uracil), reactive metabolites, chemicals in the environment and radiation.


DNA REPAIRBase excision repair: specific base

change • DNA glycosylase: recognise a specific

type of altered base in DNA and catalyse its hydrolytic removal, including: those that remove deaminated Cs, deaminated As, different types of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon–carbon double bond has been accidentally converted to a carbon–carbon single bond

• AP endonuclease: cut the sugar backbone and add in nucleotides. Depurination can be therefore directly repaired by AP endonuclease.


DNA REPAIR

Nucleotide excision repair: distortion in double helix

• Excision nuclease finds out distortion in double helix, including covalent reaction between DNA bases and large hydrocarbons and base dimer (T-T,C-T,C-C)

• DNA helicase cuts the section of distorted DNA out

• DNA polymerase rebuilds the double helix and DNA ligase ligates the helix


DNA REPAIRTranslesion DNA polymerase is used in emergencies for replication highly-damaged DNA sequences. However, they are not accurate as DNA polymerase due to lack of exonucleolytic proofreading activity. They are only released in emergencies and make “good guesses”.

http://sites.udel.edu/zhuanggroup/2015/03/19/molecular-mechanism-of-dna-translesion-synthesis/

DNA REPAIR

Transcription-coupled excision repair: Nucleotide excision repair protein couples with RNA polymerase which ensures the vital sections of genes are correct in gene expression

Friedberg, Errol C. "How Nucleotide Excision Repair Protects against Cancer." Nature Reviews Cancer Nat Rev Cancer 1.1 (2001): 22-33.

Web.

DNA REPAIRNonhomologous end joining:• broken ends are simply brought together

by DNA ligation• quick and dirty• deletion of DNA sequences occur at the

site of ligation and will lead to loss of nucleotides

• small amount of nucleotides loss is acceptable in mammalian somatic cells due to a large genome

• mistakes can happen: broken chromosomes mistakenly covalently attach to another


HOMOLOGOUS RECOMBINATIONHomologous recombination:• accurately correct double stranded break• occurs just after DNA replication, when the

two daughter DNA molecules lie close together and one can serve as a template for repair of the other.

• 5’ end of the damaged DNA is digested by specialised nuclease to produce overhanging single-strand 3’ end

• Strand exchange: one of the single-strand 3ʹ ends from the damaged DNA molecule worms its way into the template duplex and searches it for homologous sequences through base-pairing.

• An accurate DNA polymerase extends the invading DNA strand using the information form the undamaged strand.

• DNA synthesis continues using the strands from damaged DNA as templates

• DNA ligation to form complete double helix.


HOMOLOGOUS RECOMBINATION


HOMOLOGOUS RECOMBINATIONStrand Exchange:• special protein does this job, in E Coli. is RecA and in all eukaryotes is

Rad51• RecA first binds cooperatively to the invading single strand, forming a

protein–DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out

• This unusual protein–DNA lament then binds to duplex DNA in a way that stretches the duplex, destabilizing it and making it easy to pull the strands apart.

• The invading single strand then can sample the sequence of the duplex by conventional base-pairing. This sampling occurs in triplet nucleotide blocks: if a triplet match is found, the adjacent triplet is sampled, and so on.


Ragunathan, Kaushik, Chirlmin Joo, and Taekjip Ha. "Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution." Structure 19.8 (2011): 1064-073. Web. http://lion.freeoda.com/files/page_666.html

http://lion.freeoda.com/files/page_666.html

HOMOLOGOUS RECOMBINATIONHomologous Recombination is crucial for meiosis:• programmed double stranded break is preformed by a specialised protein

(Spo11 in budding yeast). Like a topoisomerase, Spo11 remains on the broken DNA sequence

• Specialised nuclease chews back at the 5’ end of the double helix, degraded the Spo11 and leaving a overhanging 3’ end

• Holiday junction (cross-strand exchange) is formed, two double-strand DNA helixes are connected with specific protein, thereby stabilises the open symmetric isomers.

http://www.jbsdonline.com/molecular-dynamics-simulations-dna-holliday-junctions-conformational-stability-and-transitions-p18150.html



HOMOLOGOUS RECOMBINATIONSpecialised proteins that bind to the holiday auctions can catalyse a reaction known as branch migration, whereby DNA is spooled through the holiday auction by continually breaking and reforming.Holiday auction therefore can move and expand the region of heteroduplex DNA from initial site using the energy from ATP


The outcome of the holiday junction can be non-crossover or crossover. 90% of homologous recombination is non-crossover. But the crossover has significant meanings.Crossover in one position will inhibit crossover in the neighbouring regions. Crossover control ensures the roughly even distribution of crossover points along the chromosomes.Roughly two crossovers occur per chromosome per mitosis


Gene conversion: If the two strands that make up a heteroduplex region do not have identical nucleotide sequences, mismatched base pairs are formed, and these are often repaired by the cell’s mismatch repair system. However, the mismatch repair system cannot distinguish between the paternal and maternal strands and will randomly choose the strand to be used as a template. As a consequence, one allele will be lost and the other duplicated, resulting in net “conversion” of one allele to the other.


TRANSPOSITIONMobile genetic element: a wide variety of specialised segments of DNA that can be moved from one position in a genome into anotherMobile elements that move by the way of transposition are called transposons, or transposable elements

TRANSPOSITIONIn transposition, a specific enzyme, usually encoded by the transposon itself and typically called a transposase, acts on specific DNA sequences at each end of the transposon, causing it to insert into a new target DNA site.

Most transposons move very rarely, in bacteria, transposons move once per 105 cell divisionMore frequent movement will probably destroy the cell genome.Transposons can be classified into DNA-only transposons, retroviral-like retrotransposons, nonretroviral retrotransposons.

https://www.ucsf.edu/news/2013/02/13535/gene-invaders-are-stymied-cells-genome-defense

https://www.ucsf.edu/news/2013/02/13535/gene-invaders-are-stymied-cells-genome-defense

TRANSPOSITIONDNA-only transposon: they exist only as DNA during their movement, predominate in bacteria and they are largely responsible for the spreading of antibiotic resistance.DNA-only transposon can be relocated from the donor site to the target site by cut-and-paste transposition. This reaction produces a short duplicated of the target DNA sequence at the insertion site, which makes transposon inserted and ligated perfectly to the insertion site. At both ends of transposon, short inverted repeat sequence are found to indicated its identity.Double-stranded break cause by the loss of transposons can be repaired either by homologous recombination or non-homologous end joining which will leaves a mutation at the original transposon site.

TRANSPOSITION


TRANSPOSITION Certain viruses are considered mobile genetic elements because they use transposition mechanism to integrate their genomes into that of their host cell.Retrovirus: exists as a single-stranded RNA genome packed into a protein shell along with a virus-encoded reverse transcriptase enzymeThe infection procedures of retrovirus involves turning single-stranded RNA into double stranded DNA by reverse transcriptase, then virus-encoded transposase called integrase inserts the viral DNA into the chromosome by a cut-and-paste transposition.

TRANSPOSITION


TRANSPOSITION Retroviral-like retrotransposons is relocated like retrovirus but lack of the protein coat.The first step in their transposition is the transcription of the entire transposon, producing an RNA copy of the element that is typically several thousand nucleotides long. This transcript, which is translated as a messenger RNA by the host cell, encodes a reverse transcriptase enzyme. This enzyme makes a double-strand DNA copy of the RNA molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus. Then, the linear double-stranded DNA is inserted into the chromosome by intergrase.

TRANSPOSITION Nonretroviral retrotransposon: distinct mechanism requires a complex of endonuclease and reverse transcriptase


CONSERVATIVE SITE-SPECIFIC RECOMBINATIONBreaking and rejoining DNA sequence at two specific site.Depending on the position and orientation, it can be classified into DNA integration, DNA excision and DNA inversion


CONSERVATIVE SITE-SPECIFIC RECOMBINATIONTransposition Conservative site-specific

recombinationrequires only that the transposon have a specialized sequence

requires specialized DNA sequences on both the donor and recipient DNA

does not proceed through a covalently joined protein–DNA intermediate

recombinases that catalyze conservative site-specific recombination resemble topoisomerases in the sense that they form transient high-energy covalent bonds with the DNA and use this energy to complete the DNA rearrangements

leaves gaps in the DNA that must be repaired by DNA polymerases.

No gaps

CONSERVATIVE SITE-SPECIFIC RECOMBINATIONConservative site-specific recombination can be also used in control of gene expression.Gene inversion can change the orientation of the promoter genes and therefore change the gene expression. Due to reversibility, the gene on the both side and be switch on and off easily.


THE END

dna replication, repair and recombination

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