Dna Damage and Repair - Lecture notes, lectures 1 - 3 PDF

Preview

Summary

Download Dna Damage and Repair - Lecture notes, lectures 1 - 3 PDF

Description

DNA Damage and Repair Types of DNA Damage: 1. Replication errors Repair Pathway followed: MMR 2. Base tautomers ♥ Base tautomerism may result in either Transition or Transversion ♥ Transition: GC - AT; AT - GC (Order of Purines and Pyrimidine is conserved) ♥ Transversion: GC - TA or CG; AT - CG or TA (Order of Purines and Pyrimidines is reversed) Cytosine Deamination ♥ Cytosine in DNA spontaneously deaminates to form Uracil. ♥ Deamination of Cytosine is mutagenic because Uracil pairs with Adenine and so one of the daughter strands will contain a U-A base pair rather than the original C-G base pair. 3. Covalent modification  Oxidative Damage Oxidative damage to Guanine ♥ Mutagens include reactive oxygen species, such as hydroxyl radical. ♥ Hydroxyl radical reacts Guanine to form 8-Oxoguanine. ♥ 8-Oxoguanine is mutagenetic because it often pairs with Adenine rather than Cytosine in DNA replication. ♥ Repair Pathway followed: BER        



UV-damage (Cytosine) Ultraviolet component of sunlight is a ubiquitous DNA-damaging agent Major effect is to covalently link adjacent pyrimidine residues along a DNA strand. Such a pyrimidine dimer cannot fit into a double helix, and so replication and gene expression are blocked until the lesion is removed. Example of an Intrastrand Cross-link is a Thymine Dimer. Both the participating bases are in the same strand of the double helix. Cross-links between bases on opposite strands can also be introduced by various agents. Psoralen (Compounds produced by a Chinese herb), forms such Interstrand Cross-Links. Interstrand cross-links disrupt replication since they prevent strand separation. ♥ Repair Pathway followed: Direct Reversal, NER Chemical Damage (including alkylation) Deamination of Adenine ♥ Adenine can also be deaminated by Nitrous Acid (HNO2) to form Hypoxanthine. ♥ The process is mutagenic because Hypoxanthine pairs with Cytosine rather than Thymine. ♥ Repair Pathway followed: BER Alkylation ♥ Nucleotide bases are subject to Alkylation. ♥ Electrophilic centers can be attacked by nucleophiles such as N-7 of Guanine and Adenine to form alkylated adducts.

♥ Some compounds are converted into highly active electrophiles through the action of enzymes that normally play a role in detoxification. ♥ Example: Aflatoxin B1 is a compound that is produced by mould on peanuts and other foods. ♥ Cytochrome p450 enzyme converts this compound into a highly reactive Epoxide which then reacts with N-7 atom of Guanosine to form a mutagenic adduct that frequently leads to a G-C to T-A transversion. ♥ Repair Pathway followed: Direct Reversal 

Electromagnetic radiation damage ♥ High-energy electromagnetic radiations such as X-rays can damage DNA by producing high concentrations of reactive species in solution, ♥ X-ray exposure can induce DNA damages likes single- and double-stranded breaks in the DNA. ♥ Repair Pathway followed: Non-homologous end joining

4. Non-covalent Interactions

DNA Repair Mechanisms ♥ Many systems repair DNA by using sequence information from the uncompromised strand. ♥ Such single-strand replication systems follow a similar mechanistic outline: A. Recognize the offending base(s) B. Removal of the offending base(s) C. Repair the resulting gap with a DNA Polymerase and DNA Ligase.

Types of Repair Mechanisms: 1. Direct Repair ♥ Example: Photochemical cleavage of Pyrimidine Dimers. ♥ All cells contain a Photoreactivating Enzyme called DNA Photolyase. ♥ The E.Coli enzyme contains bound N^5,N^10-methenyltetrahydrofolate and Flavin Adenine Dinucleotide (FAD) cofactors. ♥ The enzyme binds to the distorted region of DNA. ♥ Using light energy - specifically, absorption of a photon by N^5,N^10methenyltertahydrofolate coenzyme leads to the formation of an excited state that cleaves the dimer into its component bases. 2. Base Excision Repair ♥ Involves the excision of modified bases such as 3-methyladenine by the E.Coli enzyme, AlkA. ♥ Binding of the enzyme to damaged DNA, flips the affect base out of the DNA double helix and into the active site of the enzyme. ♥ Enzyme then acts as a Glycosylase, cleaving the Glycosidic Bond to release the damaged base. ♥ At this stage, the DNA backbone is intact but it missing a base - This hole is known as the AP Site because it is either Apurinic (Devoid of A or G) or Apyrimidinic (Devoid of C or T). ♥ An AP Endonuclease recognizes this defect and nicks the backbone adjacent to the missing base.

♥ Deoxyribose Phosphodiesterase excises the residual deoxyribose phosphate unit, and DNA Polymerase I inserts an undamaged nucleotide as dictated by the base on the undamaged complementary strand. ♥ The repaired strand is finally sealed by DNA Ligase. 3. Nucleotide Excision Repair (NER) ♥ Utilized best for the excision of Pyrimidine Dimers. ♥ 3 enzymatic activities are essential for this repair process. ♥ First, an enzyme consisting of the proteins encoded by the UvrABC genes detect the distortion produced by the DNA damage. ♥ UvrABC enzyme then cuts the damaged DNA strand at 2 sites, 8 nucleotides away from the damaged site on the 5' side and 4 nucleotides away on the 3' side. ♥ The 12-residue oligonucleotide excised by this Excinuclease then diffuses away. ♥ DNA Polymerase I enters the cap to carry out the repair synthesis. ♥ 3' end of the nicked strand is the primer, and the intact complementary strand is the template. ♥ Finally, 3' end of the newly synthesised stretch of DNA and the original part of the DNA chain are joined by DNA ligase. 4. Mismatch Repair ♥ Consists of at least 2 proteins, 1 for detecting the mismatch and the other for recruiting an endonuclease that cleaves the newly synthesised DNA strand close to the lesion to facilitate repair. ♥ In E.Coli, these proteins are called MutS and MutL and the endonuclease is MutH. MutS recognizes and binds to the mismatch     

This mutation is prevented by a repair system that recognizes Uracil foreign to DNA. The repair enzyme, Uracil-N-Glycosylase (Type of Uracil DNA Glycosylase is homologous to AlkA. Enzyme hydrolyses the glycosidic bond between Uracil and deoxyribose moieties but does not attack thymine-containing nucleotides. AP site generated is repaired to reinsert Cytosine. Thus, the methyl group on Thymine is a tag that distinguishes Thymine from Deaminated Cytosine.

1. Initiation: Before DNA Replication begins, parental strands must be separated and stabilised transiently in the single-stranded state. After this stage, synthesis of daughter strands can be initiated at the replication fork.  Initiation of replication takes place at a fixed sequence (OriC) from which the replication forks move bidirectionally until they reach the terminal sequence terC  DnaA protein specific for initiation of replication 2. Elongation: Complex of proteins called Replisome involved in this stage. Does not exist as an independent unit but instead, forms a protein complex with the particular structure that the DNA takes up at the replication fork. As replisome moves along parental strand, DNA helix unwinds and daughter strands are synthesised. 3. Termination: Termination required following DNA Replication and separation of two identical chromosomes produced required which requires manipulation of higher order DNA structure.

DNA Replication first requires a Helicase and Single-Strand Binding Protein (SSBP)    



    

Helicase is an enzyme that separates (melts) the strands of DNA, using hydrolysis of ATP to provide the necessary energy. Helicase has one conformation that binds to duplex DNA and another conformation that binds to single stranded DNA. Alternation between the two conformations is what drives the motor to melt DNA and requires ATP hydrolysis - 1 ATP molecule is hydrolysed for each base pair that is unwound. Helicase cannot initiated unwinding of duplex DNA, it can only initiate unwinding at a singlestranded region adjacent to a duplex. SSBP binds to the single-stranded DNA, protecting it and preventing it fro reforming the duplex state. SSBP binds in a cooperative manner, binding of additional monomers to the existing complex is enhanced. E.Coli SSB - Tetramer of 74 kD, Eukaryotic SSB - Trimer Unwinding by Helicase generates 2 strands which are then bound by SSB. Significance of cooperative binding is such that binding of one protein molecule makes it much easier for another to bind. Thus, once binding reaction has started on a particular DNA molecule, it is rapidly extended until all of the single-stranded DNA is covered with SSB protein. SSB is not a DNA unwinding protein, its main function is to stabilize DNA that is already in the single-stranded condition. SSB binds as the replication fork advances, keeping the two parental strands separate so that they are in the appropriate condition to act as templates.

Following unwinding by Helicase and stabilization by SSBP, a Primer is required to start DNA Synthesis     

DNA Polymerases cannot initiate synthesis of a chain of DNA de novo, but can only elongate a chain. Synthesis of a new strand can only occur from a preexisting 3'-OH end and the template strand must be converted to a single-stranded condition 3'-OH end is called a Primer Sequence of RNA is synthesised on the template so that the free 3'-OH end of the RNA chain is extended by DNA Polymerase; OR Preformed RNA (tRNA) pairs with the template, allowing its 3'-OH end to be used to prime DNA synthesis.

   

Primer is required to provide 3'-OH end to start off the DNA chains on both leading and lagging strands. Leading Strand only requires one such initiation event, whereas, Lagging Strand requires a series of initiation events due to each Okazaki fragment requiring its own star de novo. Each Okazaki fragment starts with a primer sequence of RNA approx. 10 base pairs long that provides the 3'-OH end for extension by DNA Polymerase

Primase (dnaG) is required to catalyze the actual priming reaction 

Primase is an RNA Polymerase that is used only under specific circumstances i.e. to synthesize short stretches of RNA that are used as primers for DNA synthesis.

A Helicase is required to generate single strands, a Single-Strand Binding Protein is required to maintain the single-stranded state and the Primase synthesizes the RNA primer

DNA Polymerases i. ii. iii.

iv.     

DNA Polymerase I involved in repair of damaged DNA and in a subsidiary role of semiconservative replication DNA Polymerase II required to restart progress of replication fork when it is stopped by DNA damage DNA Polymerase III responsible for de novo synthesis of new strands of DNA. Multisubunit protein.

DNA Polymerase I: Along with the ability to synthesize DNA in 5'-3' direction, replicases also have 3'-5' nuclease activity. 3'-5' Nuclease activity required to excise bases that have been added to the DNA incorrectly Serves as a 'proofreading' mechanism. Single polypeptide of 103 kD, chain can be cleaved into two parts by proteolytic treatment. Larger cleavage product - Klenow fragment (68 kD) - possesses polymerase and proofreading activities. Smaller cleavage product (35 kD) possesses 5'-3' exonucleolytic activity. Excises small groups of nucleotides (approx. 10 bases at a time). Coordinated with proofreading activity.

A. Nick Translation Function of DNA Polymerase I:  Unique ability of the enzyme to start replication at a nick in the DNA.  At a point where a phosphodiester bond has been broken in a double stranded DNA, enzyme extends the 3'-OH end.  As the new segment of DNA is synthesized, it displaces the existing homologous strand in the duplex.  Displaced strand is degraded by the 5'-3' exonucleolytic activity of the enzyme.  Properties of DNA remain unaltered, except that a segment of the DNA has been replaced by a newly synthesized segment and the nick has been moved further along the duplex.  Coupled 5'-3' synthetic/3'-5' exonucleolytic activity is used most extensively to fill in short singlestranded regions in double-stranded DNA.  Such regions arise during lagging strand DNA replication.

     

DNA Polymerase Common Structure: Enzyme structure divided into independent domains described using the analogy of a human right hand. DNA binds in a large cleft of the 3 domains. "Palm" of the enzyme contains conserved sequence motifs which provide the catalytic active site "Fingers" allow for correct positioning of the parent template at the active site. "Thumb" binds the DNA as it exists the enzyme, and is important in processivity. Exonuclease activity of the enzyme exists on an independent domain with its own catalytic site.

Leading and Lagging Strands    

   

 



 

Problem posed by structure of DNA: Antiparallel structure of the two strands of duplex DNA pose a problem for replication and Coiling of the two strands around each other As the replication fork advances, daughter strands must be synthesised on both of the exposed parental single strands. Fork template strand moves in 5'-3' direction on one strand and 3'-5 direction on the other strand. Since overall direction of synthesis must remain 5'-3', the problem is solved by synthesizing the new strand on the 5' to 3' template in short fragments/each synthesized in the backwards direction. Leading Strand (Forward Strand): DNA synthesis can proceed continuously from 5'-3' direction as the parental DNA duplex is unwound. Each new DNA strand, leading and lagging strand is synthesized by an individual catalytic unit. One enzyme unit is moving in the same direction as the unwinding point of the replication fork and synthesizing the leading strand continuously Lagging Strand (Backward Strand): Stretch of single-stranded parental DNA must be exposed, the daughter strand is then synthesised in the reverse direction relative to fork movement. A series of fragments (Okazaki fragments-Found in prokaryotes and eukaryotes) are synthesised in the 5'-3' direction and are then joined together to create an intact lagging strand - Semi discontinuous Replication. The other enzyme unit is moving backward relative to the DNA. When synthesis of one Okazaki fragment is completed, synthesis of the next Okazaki fragment is required to start at a new location approximately in the vicinity of the growing point for the leading strand. This requires DNA Polymerase III to disengage from the template and reconnect to the template at a new location at a primer to start a new Okazaki fragment. As the Replisome (DNA Pol III) moves along DNA, unwinding the parental strands, one enzyme unit elongates the leading strand. Periodically, the Primosome activity initiates an Okazaki fragment on the lagging strand and the other enzyme unit must then move in the reverse direction to synthesize DNA.

DNA Polymerase III (Replisome) Holoenzyme and its Sub complexes  

Three-polymerase core structure i.e. 2 Pol III catalytic cores responsible for synthesis of lagging strand and 1 Pol III catalytic core responsible for leading strand. Holoenzyme is a complex of 900 kD that contains 10 different proteins organized into 4 types of subcomplex:



 

   

     

Each catalytic core consists of an Alpha subunit which carries out DNA Polymerase activity, E subunit that carries out 3'-5' exonuclease activity and the Theta subunit which stimulates exonuclease There are 2 copies of the dimerizing subunit (Pi) which link the 2 catalytic cores together and maintain dimeric structure. There are 2 copies of the Beta clamp which is responsible for holding catalytic cores onto their template strands. Each clamp consists of a homodimer of B subunits, B2 ring, which bind around the DNA and ensures processivity. B is strongly bound to DNA but provides the sliding clamp that allows the holoenzyme to slide along the duplex molecule. Ring shaped dimer is formed is formed around DNA and the space between the protein ring and DNA is fill by water. Structure explains high processivity - The enzyme can transiently dissociate but does not fall off and diffuse away. Alpha helices on the inside of the Beta clamp have some positive charges that may interact with DNA via the water molecules. Loading of Beta Clamp: The clamp is a circle of subunits surrounding the DNA, its assembly or removal requires the use of an energy dependent process by the clamp loader. The Gamma complex is a group of 7 proteins, encoded by 5 genes that comprise the clamp loader - Clamp loader cleaves ATP to load the clamp on DNA. Gamma clamp loader is a pentameric circular structure that binds an open form of the B2 ring preparatory to loading it onto DNA. Binding of Gamma to the ring, destabilizes it and opens it, facilitated by ATP. While one polymerase subunit of DNA Polymerase synthesizes the leading strand continuously, the other cyclically initiates and terminates the Okazaki fragments of the lagging strand. Replication fork created by Helicase which forms a hexameric ring - Translocates in the 5'-3' direction on the template for the lagging strand. Helicase is connected to the two DNA Polymerase catalytic units each of which is associated with a sliding clamp.

Mechanism and Structure of DNA Polymerase III     

i. ii. 

A catalytic core is associated with each template strand Holoenzyme moves continuously along the template for the leading strand Template for the lagging strand is 'pulled through' thus creating a loop in the DNA. DnaB contacts the Pi subunits of the clamp loader. A direct connection is established between the helicase-primase complex and the catalytic cores. There are two effects of the links: Increase in the speed of DNA synthesis by increasing the rate of movement by DNA Polymerase tenfold. Prevent the leading strand polymerase from falling off, i.e. to increase processivity. Synthesis of leading strand creates a single stranded DNA loop which proves the template for lagging strand synthesis.

On completion of Okazaki fragment  

All components of the replication apparatus function processively except Primase and B2 clamp. B2 clamp must be cracked open by the Gamma clamp loader when the synthesis of each fragment is completed.

  

Clamp loader causes B2 clamp to alter its conformation to an unstable configuration, which then springs open. New B2 clamp is recruited by the clamp loader to initiate the next Okazaki fragment. Lagging strand polymerase transfers from one B2 clamp to the next in each cycle, without dissociating from the replicating complex.

Okazaki Fragments are linked by DNA Ligase 

A. B. C. D. E.

Process involves: Synthesis of RNA Primer Extension with DNA Removal of RNA primer Replacement by a stretch of DNA Covalent linking of adjacent Okazaki fragments.

In mammalian systems, Okazaki fragments are connected in a 2 step process: 1. Synthesis of Okazaki fragment displaces the RNA Primer of the preceding fragment in the form of a flap.  Primer is cleaved by enzyme FEN1 (f lap endonuclease 1) 

2. Once the RNA has been removed and replaced, the adjacent Okazaki fragments must be linked together.  3'-OH end of one fragment is adjacent to the 5'-phosphate end of the previous fragment.  Enzyme DNA Ligase make a bond by using a complex with AMP.  AMP of the enzyme complex becomes attached to the 5' phosphate of the nick and then a phosphodiester bond is formed with the 3'-Oh terminus of the nick,  Releasing the enzyme and the AMP.

Topoisomerases  

  

    

Isomerase enzymes that regulate the overwinding or underwinding of DNA and act on the topology of DNA During DNA Replication and Transcription, DNA becomes overwound ahead of a replication fork , this tension would eventually stop the ability of the enzymes involved in these processes to continue down the DNA strand. They relieve the super he...