Lecture 4: Replication Errors and Repair System - Molecular Diagnostics

MOLECULAR DIAGNOSTICS Lecture 4 Replication Errors and Repair System Cell Cycle Checkpoints and Genomic Diversity (SNP) DNA DAMAGE AND REPAIR http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNArepair.html CAUSES OF DNA DAMAGES TYPES OF DNA DAMAGES AGENTS THAT DAMAGE DNA • Certain wa...

MOLECULAR DIAGNOSTICS Lecture 4 Replication Errors and Repair System Cell Cycle Checkpoints and Genomic Diversity (SNP) DNA DAMAGE AND REPAIR http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNArepair.html CAUSES OF DNA DAMAGES TYPES OF DNA DAMAGES AGENTS THAT DAMAGE DNA • Certain wavelengths of radiation • ionizing radiation such as gamma rays and X-rays • ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield. • Chemicals in the environment • many hydrocarbons, including some found in cigarette smoke • some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts • Chemicals used in chemotherapy, especially chemotherapy of cancers • Intrinsic Spontaneous mutation • most error during DNA replication by error of polymerase 3’ to 5’ exonuclease activity • MMR enzyme mutation caused mismatch repair failure • Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways. REPAIRING DAMAGED BASES • The recent publication of the human genome has revealed 130 genes whose products participate in DNA repair. More is expected to be discovered • Damaged or inappropriate bases can be repaired by several mechanisms: • Direct chemical reversal of the damage • Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes. • Base Excision Repair (BER) • Nucleotide Excision Repair (NER) • Mismatch Repair (MMR) • The 2015 Nobel Prize in chemistry was shared by three researchers for their pioneering work in DNA repair: Tomas Lindahl (BER), Aziz Sancar (NER), and Paul Modrich (MMR). • Good review: http://www.nature.com/nrm/journal/v9/n4/full/nrm2351.html DIRECT REPAIR GENES • DNA photolyase • Natural repair system for pyrimidine dimers caused by UV damage • Directly reverse cyclobutane pyrimidine dimer (CPD) via photochemical reactions • O6-methylguanine-DNA methyltransferase (MGMT) • naturally occurring mutagenic DNA lesion O6-methylguanine back to guanine • prevents mismatch and errors during DNA replication and transcription • the methylation state of the MGMT gene promotor determined whether tumor cells would be responsive to temozolomide drug therapy BASE EXCISION REPAIR (BER) • The damaged base estimated to occur some 20,000 times a day in each cell in our body! • Remove it by a DNA glycosylase. There are at least 8 genes encoding different DNA glycosylases. • Each enzyme responsible for identifying and removing a specific kind of base damage. • Two genes encoding enzymes (AP endonuclease and DNA Exonuclease) function to removal deoxyribose phosphate in the backbone, producing a gap. • Replacement with the correct nucleotide. This relies on DNA polymerase beta, one of at least 11 DNA polymerases encoded by our genes. • Ligation of the break in the strand. Two enzymes are known that can do this; both require ATP to provide the needed energy. BASE EXCISION REPAIR • One stand of DNA contains deaminated base, such as Uracil • DNA glycosylases scans the DNA and removes Uracil, leaving AP site • AP endonuclease locates AP site and nicks backbone • DNA Exonuclease removes nucleotides near the nick, leaving gap • DNA Polymerase synthesizes to fill in gap • DNA Ligase seals the backbone NUCLEOTIDE EXCISION REPAIR (NER) • NER differs from BER in several ways. • It uses different enzymes (XP products). • Even "though there may be only a single "bad" base to correct, NER removes a large "patch around the damage such as to removes DNA damage induced by ultraviolet light (UV), such as thymine dimer. • The steps and some key players: • The damage is recognized by Transcription Factor IIH, (TFIIH also functions in normal transcription) and may be more protein factors that assemble at the location. • The DNA is unwound producing a "bubble". The enzyme system (Numerous proteins, including XP products (XPA, XPB, XPF, XPG), make cut both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed. • A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct nucleotides with DNA polymerase delta and epsilon. • A DNA ligase covalently inserts the fresh piece into the backbone. THYMINE DIMER XERODERMA PIGMENTOSUM (XP) • A rare inherited autosomal recessive disease of humans in which a deficiency of excinuclease occurs • XP can be caused by mutations in any one of several genes (XPA, XPB, XPF, XPG), all of which have roles to play in NER • XP resulting in skin discolouration and multiple tumours on exposure to UV light. • Unrepaired pyrimidine dimers in humans may lead to melanoma MISMATCH REPAIR (MMR) • DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage • Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A•T, C•G) • It can enlist the aid of enzymes involved in both base-excision repair (BER) and nucleotideexcision repair (NER) as well as using enzymes specialized for this function. • Recognition of a mismatch requires several different proteins including one encoded by MSH2 known as MutS protein homolog 2, a caretaker gene • Cutting the mismatch out also requires several proteins, including one encoded by MLH1 known as MutL homologs. It forms a complex with MutS and MutH, increasing the MutS footprint on the DNA. • Mutations in either of these genes predisposes the person to an inherited form of colon cancer. So these genes qualify as tumor suppressor genes Mismatch Repair in Prokaryotes and Eukaryotes Which strand is new and which is the parent? • • • Mut S recognizes and binds mismatch Mut L links S to H Mut H recognizes the CH3-parental strand and makes nick on daughter strand • In human: MutS = hMSH (1-6); MutL = hMLH1 and hPMS2; MutH = GTBP DISEASES CAUSED BY DEFECT OF DNA REPAIR EXAMPLES OF DNA REPAIR DEFECTS AND CANCER REPAIRING STRAND BREAKS • Single-Strand Breaks (SSBs) • Breaks in a single strand of the DNA molecule are repaired using the same enzyme systems that are used in Base-Excision Repair (BER). • BER, NER and MMR are all strand specific SSB repairing system • Double-Strand Breaks (DSBs) • There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule: • Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ). • Homologous Recombination (next page) REPAIRING OF DOUBLE STRAND BREAKS • Homologous Recombination (also known as HomologyDirected Repair HDR). • Sister chromatid (available in G2 after chromosome duplication), or • Homologous chromosome (in G1; that is, before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog - a task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by NHEJ. or on the • Same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-toback). • Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast and ovarian cancers THE CELL CYCLE CONTROLS Why Its so Important to Regulate Entry into the Cell Cycle Checkpoints Inactivation of cell cycle Adapted from Modified from: Kim Foglia, Explore Biology etc. PHASES OF THE CELL CYCLE • G0 • G1 Check point • S • G2 Checkpoint • M • prophase, • Metaphase checkpoint • anaphase, • telophase • cytokinesis FIVE PHASES OF CELL CYCLE AND THE CHECKPOINTS IN THE CYCLE State Description Abbrevia tion Gap 0 G0 A resting phase where the cell has left the cycle and has stopped dividing. Gap 1 G1 Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis. Synthesis S quiescent/ senescent Interphase Gap 2 Cell division Mitosis Functions DNA replication occurs during this phase. G2 During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide. M Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division. MAJOR PROTEINS THAT CONTROL CELL CYCLE • Control Proteins • Cyclin-dependent protein kinases (Cdks) • Cyclins • Complexes: Cdk-cyclin • ability of Cdk to “P” target is dependent on the cyclin that it forms a complex with CELL CYCLE CHECKPOINT PROTEINS • G1 checkpoint: • Active cyclin D-cdk4 complexes phosphorylate retinoblastoma protein (pRb) in the nucleus. Unphosphorylated Rb acts as an inhibitor of G1 by preventing E2F-mediated transcription. • Activated P53-p21 interaction also prevent G1 to S phase transition • G2 checkpoint • DNA damage in the G2 phase initiate a signaling cascade that regulates wee1 and cdc25 activity, therefore controlling mitotic entry via cyclin B-cdK2 can delay in mitotic entry. • Mitotic checkpoint • Spindle assembly checkpoint (SAC), through P53-cdK2-GADD45 cascade, to ensure chromosome segregation is correct. • prevents anaphase onset until all chromosomes are properly attached to the spindle. APOPTOSIS • The process of programmed cell death that may occur in multicellular organisms. • Biochemical events lead to characteristic cell changes (morphology) and death (different from necrotic or autophagic cell death): • blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. • Highly regulated and controlled process that confers advantages during an organism's lifecycle. • Apoptotic Genes • AIFM2, BAK1, BBC3, BCL2, BCL2L1 (BCL-X), BID, BNIP3, CDKN2A (p16INK4), DNM1L, MPV17, PMAIP1 (NOXA), SFN (14-3-3s), SH3GLB1, SOD2, TP53. NECROSIS VS APOPTOSIS cells die accidentally due to injury, trauma (ex. a poisonous spider bite), or lack of nutrients (ex. lack of blood supply) Cells commit suicide when lacking any incoming survival, or severe viral infection. or when they detect extensive DNA damage in their own nucleus. Cells will murder other cells to clear out unneeded cells or to eliminate potentially self-attacking immune cells. UBIQUITIN PROTEASOME PATHWAY (UPP) ENZYMES AND CYCLIN DEGRADATION Activating Conjugating Ligase INACTIVATING CYCLIN DEPENDENT KINASE (CDK) THROUGH UBIQUITINATION PATHWAY THE UBIQUITIN-PROTEASOME SYSTEM • Proteosome, a complexes inside all eukaryotes and archaea, and in some bacteria. • Main function is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds • Proteasomes are located both in the nucleus and in the cytoplasm. • The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress. • Protein degradation during anaphase of mitosis of cell cycle. • The importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways (UPP) was acknowledged in the award of the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose GENOMIC DIVERSITY Sequence Variants Single Nuclotide Polymorphism (SNP) Adapted from Andrea Ferreira-Gonzalez, Ph.D. Department of Pathology Virginia Commonwealth University HUMAN GENOME PROJECT • Launched in 1989 -expected to take 15 years • Competing Celera project launched in 1998 • Genome estimated to be 92% complete • 1st Draft released in 2000 • "Complete" genome released in 2003 • Sequence of last chromosome published in 2006 • Cost: rv $3 billion • Celera: rv $300 million THE HUMAN GENOME • All humans share 99.9% of the same genetic sequence • 90% of human genome variation comes from Single Nucleotide Polymorphisms (SNPs) • The most common sources of variation between humans are single nucleotide polymorphisms (SNPs)—single base differences between genomic sequences.” -- Aravinda Chakravarti, Nature, 409 (Feb 2001), 822-823 GENOME SEQUENCING PROJECT FINDS SNPS • The Human Genome Project involves sequencing DNA cloned from a number of different people.[The Celera sequence comes from 5 people] • SNPs occur normally throughout a person’s DNA. • It occur almost once in every 1,000 nucleotides on average, which means there are roughly 2-10 million SNPs in a person's genome. • This inevitably leads to the discovery of any sequence difference-SNP is the most common one. • SNP can act as biological markers, helping scientists locate genes that are associated with disease. • When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene’s function. https://medlineplus.gov/genetics/understanding/genomicresearch/snp POLYMORPHISMS • Most disease-causing gene mutations are uncommon in the general population. • Genetic alterations that occur in more than 1 percent of the population are called polymorphisms. • They are common enough to be considered a normal variation in the DNA. • Polymorphisms are responsible for many of the normal differences between people such as eye color, hair color, and blood type. • Although many polymorphisms have no negative effects on a person’s health, some of these variations may influence the risk of developing certain disorders. Human Genetic Variation Every human has essentially the same set of genes • But there are different forms of each gene - known as alleles – blue vs. brown eyes (or purple vs white flowers, see below) • genetic diseases such as cystic fibrosis or Huntington’s disease are caused by dysfunctional alleles • Allele for purple flowers Locus for flower-color gene Allele for white flowers Homologous pair of chromosomes LIFE CYCLE OF SNP (LONG WAY FROM MUTATION TO SNP) Appearance of new variant by mutation Survival of rare allele Increase in allele frequency after population expand New allele is fixed in population as novel polymorphism SEQUENCE VARIATION AND SNP DISTRIBUTION • Non-Coding region (2/3) • Regulatory region • Coding region (1/3) • Synonymous • Non-Synonymous • missense • Conservative • Non-Conservative • nonsense • One half of all coding sequence SNPs result in non-synonymous codon changes. LEARNING FROM OUR DIFFERENCES • Most common diseases and many drug responses have been shown to be influenced by inherited differences in our genes • Studying genetic variances can improve our understanding and treatment of disease END