Chapter 02 DNA replication and DNA Repair - Tagged.pdf

Chapter 2 DNA replication and DNA repair Dra Verónica Veses Jiménez Chapter Overview DNA replication: concept and characteristics DNA polymerases Replication in Prokaryotes Replication in Eukaryotes DNA damage and DNA repair 2 DNA replic...

Chapter 2 DNA replication and DNA repair Dra Verónica Veses Jiménez Chapter Overview DNA replication: concept and characteristics DNA polymerases Replication in Prokaryotes Replication in Eukaryotes DNA damage and DNA repair 2 DNA replication Replication is the name given to the process by which DNA is copied. This process allows the maintenance of fidelity of the genetic information of an organism. 3 Three models for replication: Semiconservative: one strand is conserved and one is synthesized de novo. Conservative: one of the daughter cells conserves all the DNA and the other one synthesizes it de novo Dispersive: both daughter cells get random pieces of DNA. 4 Replication is NOT conservative: Meselson & Stahl, 1957 5 Replication is semiconservative: 6 Replication begins at an origin and proceeds bidirectionally: Cairns, 1960’s 7 8 Replication proceeds in 5 to 3 direction and is semi-discontinuous: Okazaki, 1960s 9 DNA POLYMERASES 10 DNA is synthesized by DNA polymerases  Kornberg & col in 1955 purificated and isolated the first DNA polymerase, from E. coli, called today DNA pol I.  The fundamental reaction catalyzed is a phosphoryl group transfer. The OH at the 3’ end of the growing strand attacks the phosphorus of the incoming deoxynucleotide. The R proceed with only a minimal change in free energy, but non-covalent base-stacking and base-pairing interactions provide additional stability to the lengthened DNA product  Processivity is the average number of nucleotides added before a polymerase dissociates  Requirements of DNA pol:  Requires a template  Requires a primer 11 DNA is copied with a high degree of fidelity Replication proceeds with an extraordinary degree of fidelity. In E. coli a mistake is made only once every 109 to 1010 nucleotides. This represents 1 error every 1000-10000 replications. This relies on the correct pairing between bases and the common geometry to fit the active sit of the DNA pol. However, the high degree of accuracy requires the action of further factors. 12 Proofreading activity Almost all DNA pol include a separate 3’-5’ exonuclease activity that double-checks each nucleotide after is added. The nuclease activity removes a newly added nucleotide, being highly specific for mismatched base pairs. It does not catalyze the inverse reaction, as no pyrophosphate is involved. There is an additional mismatch repair mechanism. 13 REPLICATION IN PROKARYOTES 14 DNA polymerases (E. coli) DNA pol I: not very fast, low processivity. It does not explain replication rates by itself. mutants lacking it are still viable. Involved in replication, recombination, repair. DNA pol II: repair DNA pol III: replication DNA pol IV: repair DNA pol V: repair 15 DNA Pol I has “nick translation” activity Makes reference to the 5’-3’ exonuclease activity, that can replace a segment of DNA or RNA paired to the template strand, in a process known as nick* translation. This activity is located in structural domain that can be separated from the enzyme with mild protease treatment. * Broken phosphodiester bond 16 DNA Pol III is the replication polymerase Two core domains are linked through the tau subunits and also linked to the clamp-loading complex. Two additional subunits chi and psi are bound to the clamp- loading complex. Two beta clamps interact with the two-core subassembly, each clamp being a dimer of beta. 17 The replisome Helicases: move along DNA and separate the strands, using chemical energy from ATP Topoisomerases: relieve the topological stress created by helicases DNA-binding proteins: stabilize the separated strands Primases: synthesis of short RNA segments that act as primers DNA ligases: seal the nicks after DNA repair works done by DNA pol I 18 Phases of replication Initiation: – Replication starts at the E. coli origin, oriC. – R and I sites serve as binding sites for the initiator protein DnaA. – Region rich in A=T called the DNA unwinding element. Elongation Termination 19 Replication https://youtu.be/TNKWgcFPHqw https://youtu.be/4jtmOZaIvS0 20 Initiation Eight DnaA protein molecules, each with a bound ATP, bind at the R and I sites in the origin. The DNA is wrapped around this complex in a right handed helical structure. The DUE region is denaturated by the adjacent binding of DnaA. Hexamers of DnaB protein bind to each strain, with the aid of DnaC protein. DnaB helicase activity further unwinds the DNA in preparation for priming and DNA synthesis. 21 Elongation Leading strand: Primase (DnaG) synthesizes an RNA primer at the origin. DnaG interacts with helicase DnaB synthesizing the primer on opposite direction. Then DNA pol III keeps adding nucleotides to the primer. Lagging strand: At intervals, the primase synthesize an RNA primer for a new Okazaki fragment, each primer is then extended as far as the primer of the previously added Okazaki fragment. 22 Final steps in the synthesis of lagging strand segments RNA primers are removed by the 5’-3’ exonuclease activity of DNA polymerase I and are replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. 25 Termination The Ter sequences function as binding sites for the protein Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one direction. The other replication fork stops when it meets the arrested one. 26 REPLICATION IN EUKARYOTES 27 Replication in Eukaryotes It is coordinated with the cell cycle: regulation is mediated by the action of cyclines and cyclin- dependent kinases (CDKs). The places for the start of the replication are also rich en AT, but they vary from one replication to the next. Usually there are more than one simultaneous places of replication in each chromosome. 28 Requirements 1. Assembly of a pre-replication complex is required. 2. There are different polymerases for the nucleus and mtDNA. 3. The termination of replication requires the synthesis of specific areas: the telomeres. 29 Pre-replication complex The eukaryotic pre-RC is complex and highly regulated. In most eukaryotes it is composed of six ORC proteins (ORC1-6), Cdc6, Cdt1, and a heterohexamer of the six MCM proteins (MCM2-7). 30 Eukaryotic polymerases: Five common DNA polymerases from mammals: Alpha: nuclear, DNA replication, no proofreading Beta: nuclear, DNA repair, no proofreading Gamma: mitochondria, DNA replication, proofreading Delta: nuclear, DNA replication, proofreading Epsilon: nuclear, DNA repair (?), proofreading 31 Telomeres A telomere is a repeating DNA sequence at the end of each chromatid, which can have 15000 pairs. The function of telomeres is to prevent information loss at the ends of the chromosome. In each cell division is lost between 25 and 200 bp as the telomere reaches a minimum size "critical" chromosome is unable to replicate and the cell enters apoptosis. The activity of the telomere is regulated by two mechanisms: erosion and addition. 32 Telomerases DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication. Solution: Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of new repeats, which compensates by lengthening the chromosome. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division. 33 34 Telomerase activity The telomerase or "telomere terminal" transferases are made from RNA and protein. Its function is to stretch the chromosomes adding TTAGGG sequences at the end of chromosome. Telomerases are found in fetal tissues, adult stem cells and tumor cells. If this activity is activated in somatic cells, they become immortal, with two consequences: changes in the pattern of aging and cancer processes. 35 36 DNA REPAIR SYSTEMS Section Overview Sources of DNA damage Types of DNA damage Repair systems Diseases associated with faulty DNA repair systems 38  DNA Damage and Repair DNA in all living cells is constantly subject to damaging agents Sources of DNA damage – Natural polymerase error – Endogenous DNA damage Oxidative damage Depurination Deamination – Exogenous DNA damage Radiation Chemicals – ‘Error prone’ DNA repair – Strand breaks (double or single strand) 39  Types of DNA damage DNA Lesion Cause Incorrect base Polymerase errors Missing base Spontaneous deamination Altered base radiation Deletion/insertion Intercalating agents Linked pyrimidines (usu. T-T) UV light Single/double strand breaks Radiation, oxidative stress X-linked strands Carcinogens 40 Jan de Boer, and Jan H.J. Hoeijmakers Carcinogenesis 2000;21:453-460 41 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 42 Proofreading DNA polymerase III – proofreading polymerase – ~0.1 errors / gene In Escherichia coli, scientists have estimated an approximate mutation rate of ~1 / 1000 cells Proofreading stops most errors from occurring in the first place 43 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 44 Mismatch Repair (MMR) - I Despite extraordinary fidelity of DNA synthesis, errors do persist Such errors can be detected and repaired by the post-replication mismatch repair system Prokaryotes and eukaryotes use a similar mechanism with common structural features Defects in MMR elevate spontaneous mutation rates 10-1000x Defects in MMR underlie human predisposition to colon and other cancers 45 MMR Incorrect bases incorporated as a result of mistakes during DNA replication ( base mispairs, short insertions and deletions) are excised as single nucleotides by a group of repair proteins which can scan DNA and look for incorrectly paired bases (or unpaired bases) which will have aberrant dimensions in the double helix. Synthesis of the repair patch is done by a DNA polymerase 46 Mismatch Repair (MMR) - II Example shown here is the MutS system from E. coli. Well known and best characterised MMR system 47 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 48 Base Excision Repair For correction of specific chemical damage in DNA – Uracil (deamination of cytosine or wrongly incorporated) – Hypoxanthine (deamination of adenine) - 3-methyladenine - 7-methylguanosine - 8-oxoguanine, etc Require DNA glycosylases (to remove the damaged base) and Apurinic/Apyrimidic (AP) endonuclease (to nick DNA in the AP site created when DNA glycosylase removes the damaged base) The DNA glycosylases are specific – Uracil glycosylase – Hypoxanthine DNA glycosylase 49 Mechanism DNA glycosylase recognizes specific damaged base Cleaves glycosyl bond to remove base AP endonuclease cleaves backbone DNA Pol replaces the base 50 Different steps of BER: 1. removal of the damaged base by a DNA glycosylase. Eight enzymes, each one responsible for identifying and removing a specific kind of base damage. 2. removal of its deoxyribose phosphate in the backbone, producing a gap: an AP site. Two genes encoding enzymes with this function. 3. replacement with the correct nucleotide. Done by DNA polymerase beta (one of at least 11 DNA polymerases encoded by our genes), using the other strand as a template. 4. ligation of the break in the strand. Two enzymes are known that can do this. 51 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 52 Nucleotide Excision Repair Recognizes bulky lesions that block DNA replication (lesions produced by carcinogens) – example: UV pyrimidine photodimers. The process of NER is biochemically complicated, 30 distinct proteins that function as a large complex called the nucleotide excision repairosome. It is the most important DNA repair pathway, and the sole repair system for bulky DNA lesions, which creates a block to DNA replication and transcription. It can also repair many of the same defects that are corrected by direct repair, base excision and mismatch repair. Defects in NER underlie Xeroderma pigmentosum, Cockayne Syndrome and Trichothiodystrophy. 53 Steps in NER are: Recognition of damage by one or more protein factors. Assembly of repair complex: nucleotide excision repairosome. Double incision of the damaged strand several nucleotides away from the damaged site, on both sides, by an endonuclease. Removal of the short segment ( about 24 to 32 nucleotides) containing the damaged region, by an exonuclease. Filling in of the resulting gap by a DNA polymerase: synthesizes DNA using the opposite strand as a template Ligation: a DNA ligase binds the synthesized piece into the backbone. 54 55  Comparison of the NER system in E. coli and humans Function E. coli Humans DNA scanning UvrA XPA, XPC; RPA Nucleases UvrB, UvrC XPF, XPG Helicase UvrD XPB, XPD Replication system DNApol I/II; ligase DNApol d/e; ligase 56 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 57 Recombination DNA double-strand breaks (DSBs) are the most hazardous lesions arising in the genome of eukaryotic organisms, and yet occur normally during DNA replication, meiosis, and immune system development. The efficient repair of DSBs is crucial in maintaining genomic integrity, cellular viability, and the prevention of tumor genesis. 58 Types of recombination 59 Homologous recombination DSBs are repaired by recombination of the broken strands with their homologous sister strands during cell division. The broken strands invade the DNA structure of the ‘good’ strands, followed by new DNA synthesis and migration of the repaired strands back out of the sister DNA. 60 Cellular mechanisms to repair DNA damage Natural polymerase error – Proofreading – Mismatch repair Endogenous / Exogenous DNA damage – Base excision repair – Nucleotide excision repair – Recombination (homologous/ non-homologous end joining) – Polymerase bypass 61 Polymerase bypass Replication-blocking lesions such as UV photodimers can be repaired by NER but pose a serious problem if they are in ssDNA As a last resort, cells employ “bypass” polymerases with loosened specificity In E. coli an example is DinB (Pol IV) and homologs exist in eukaryotes; mutated in Xeroderma pigmentosum These polymerases are “error-prone” and are responsible for UV-induced mutation Expression and function highly regulated: dependent on DNA damage 62 Characteristics of lesion-bypass polymerases Error rate 100-10,000 x higher on undamaged templates Lack 3’ to 5’ proofreading exonuclease activity Support trans-lesion DNA synthesis in vitro 63 DISEASES ASSOCIATED WITH DEFECTS IN DNA REPAIR SYSTEMS 64 Xeroderma pigmentosum Autosomal recessive mutations in NER system genes. Extreme sensitivity to sunlight. Predisposition to skin cancer (mean age of skin cancer is 8 years versus 60 for normal population). 65 Cockayne Syndrome Cockayne syndrome (CS) is characterized by additional symptoms such as short stature, severe neurological abnormalities caused by demyelization, bird-like faces, tooth decay, and cataracts. CS patients have a mean life expectancy of 12.5 years. CS cells are deficient in transcription-coupled NER but are proficient in global genome NER 66 Trichothiodystrophy A third genetic disease characterized by UV sensitivity, trichothiodystrophy (TTD, literally: “sulfur-deficient brittle hair”), was reported by Price in 1980. In addition to symptoms shared with CS patients, TTD patients show characteristic sulfur-deficient, brittle hair and scaling of skin. This genetic disorder is now known to correlate with mutations in genes involved in NER (transcriptional transactions rather than regular defect in DNA repair). 67  Fanconi anemia Affects many parts of the body: bone marrow failure, physical abnormalities, organ defects, increased risk of certain cancers, with a prevalence of 1 in 160,000 individuals worldwide. Mutations in at least 15 genes can cause Fanconi anemia although 80 to 90% of cases are due to mutations in FANCA, FANCC or FANCG. Proteins produced from these genes are involved in interstrand cross-links DNA damage reparation pathway. 68 Bloom syndrome Patients have short stature, sun-­sensitive skin changes (dilated blood vessels and reddening in the skin), an increased risk of cancer (early in life), diabetes, chronic obstructive pulmonary disease and recurrent infections. Distinctive facial features and learning disabilities. Caused by mutations in the BLM gene. This gene provides instructions for making RecQ helicases: enzymes that unwind DNA. Cancer results from genetic changes that allow cells to divide in an uncontrolled way. Altered BLM protein activity may also lead to an increase in cell death, resulting in slow growth in affected individuals. 69 Hereditary nonpolyposis colon cancer (HNPCC) or Lynch syndrome Inherited disorder that increases the risk of many cancers: particularly colorectal but also small intestine, liver, gallbladder ducts, upper urinary tract, brain, skin, ovaries and uterus. Variations in the MLH1, MSH2, MSH6, PMS2 (genes involved in DNA reparation). Not all people who inherit mutations in these genes will develop cancer. Lynch syndrome cancer risk is inherited in an autosomal dominant pattern (one inherited copy of the altered gene is sufficient to increase cancer risk). 3-­‐5% of diagnosed colorectal cancer. 70 References David L. Nelson, 2012. Lehninger Principles of Biochemistry. 6th Edition. W.H. Freeman. Bruce Alberts, 2012. Molecular Biology of the Cell, 5th Edition. 5th Edition. Garland Science. https://revistageneticamedica.com/blog/elizabeth- blackburn/ Video on DNA replication. Cold Spring Harbor. Jan de Boer, and Jan H.J. Hoeijmakers, Carcinogenesis 2000;21:453-460 Cleaver JE et al. A summary of mutations in the UV- sensitive disorders. Hum Mutat 1999; 14:9-22 71