DNA Replication, Damage & Repair 2025 PDF

WE MAKE DOCTORS DNA replication, Damage and Repair Ana Gabriela Colima Fausto, PhD Learning objectives Outline the characteristics and steps involved in DNA replication Identify the function of the enzymes involved in DNA replication Describe the types of mutagens Understand th...

WE MAKE DOCTORS DNA replication, Damage and Repair Ana Gabriela Colima Fausto, PhD Learning objectives Outline the characteristics and steps involved in DNA replication Identify the function of the enzymes involved in DNA replication Describe the types of mutagens Understand the DNA repair processes that are activated in the cells Identify disorders produced by abnormalities in DNA replication: Xeroderma Pigmentosum and Fanconi Anemia. DNA REPLICATION The replication or copying of cellular DNA occurs during the S or synthesis phase of the cell cycle Process to ensure that the instructions in DNA are faithfully passed on to the newly produced cells DNA REPLICATION The double-stranded DNA of the chromatin must first unwind. Once unwound, both strands of DNA are copied simultaneously. This process requires proteins to break open the double-stranded DNA, forming a replication fork. The main enzyme that catalyzes the formation of new DNA strands is DNA polymerase DNA REPLICATION During replication, DNA polymerases select the nucleotide that is to be added to the 3′-OH end of the growing chain and catalyze the formation of the phosphodiester bond. The substrates for DNA polymerases are the four deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and a single-stranded template DNA. DNA REPLICATION DNA polymerases cannot start synthesis of a complementary strand of DNA DNA primase, synthesizes short stretches of RNA that are complementary and antiparallel to the DNA template. The RNA primer is later removed. The sequence of nucleotides that are added is dictated by the base sequence of the template (or coding) strand with which the incoming nucleotides are paired  DNA replication characteristics Semiconservative with respect to parental strand: original strand of DNA is distributed to each daughter duplex in combination with a newly synthesized strand with an antiparallel orientation. Each of the two daughter strands has half new DNA and half old DNA.  DNA replication characteristics Bidirectional with multiple origins of replication: is bidirectional and starts in several separate locations at once (DNA is copied at about 50 bp per second). As replication nears completion, “bubbles” of newly replicated DNA come together forming two new molecules Semidiscontinuous A new strand of DNA is always synthesized in the 5′ to 3′ direction. Because the two strands of DNA are antiparallel, the strand being copied is read from the 3′ end toward the 5′ end. All DNA polymerases function in the same manner: They “read” a parental strand 3′ to 5′ and synthesize a complementary antiparallel new strand 5′ to 3′. Because parental DNA has two antiparallel strands, the DNA polymerase synthesizes one strand in the 5′ to 3′ continuously. This strand is called the leading strand. The other new strand is synthesized 5′ to 3′, but discontinuously, creating fragments (Okazaki fragments that ligate (join) together later. This strand is called the discontinuous or lagging strand. Leading vs Lagging strands AMBOSS GmbH.DNA replication.https://amboss.com/. Accessed January 8th, 2025.  DNA REPLICATION This process involves proteins to break the double-stranded DNA, keep the DNA structure open, synthesize a new strand, and, finally, put them together as one long linear DNA. Other proteins are needed to remove torsion that can occur when opening a double helix.  POLYMERASE Some DNA polymerases have 3′ to 5′ exonuclease activity, or proofreading ability, that allows them to remove nucleotides that are not part of the double helix. The enzyme removes mismatched residues, thus performing an editing function. This activity enhances the fidelity of DNA replication by rechecking the correctness of base pairing before proceeding with polymerization  ENZYMES IN DNA REPLICATION Helicase Primase SSBP Ligase  Topoisomerase This underwound condition (negative supercoils) facilitates the unwinding of the double helix during replication and transcription. As the replication fork moves along the helix, rotation of the daughter molecules around one another causes the DNA strands to become overwound. The super twisting of DNA can be removed by enzymes known collectively as topoisomerases. These enzymes relieve torsional stress in DNA by inducing reversible single-stranded breaks in DNA. Telomerase Telomeres, shortens with every cell division. They protect chromosomes from degradation. Telomeric DNA consists of a tandem array of very simple sequence of DNA (in humans, it is TTAGGG). The telomerase ribonucleoprotein complex contains an RNA template, which is an integral component of the enzyme. With the help of its RNA template, it adds a series of DNA repeats to the leading strand. This addition allows the lagging strand to be completed by DNA polymerase Clinical application: Nucleoside analogs Pharmacological class of compounds with cytotoxic, immunosuppressive, and antiviral properties. They have a structural similarity to guanosine. Act as DNA polymerase inhibitors, reducing DNA synthesis and viral replication. Acyclovir and valacyclovir cause chain termination when incorporated into the DNA strand, as they lack the 3’-OH group that attaches to the next nucleoside. Penciclovir has a 3’-OH group do not cause chain termination and instead inhibit DNA elongation. DNA DAMAGE DNA damage can result from both endogenous and exogenous causes. Most DNA damage is repaired before DNA is replicated. Mutagenic agents (ones that induce mutations) are therefore most effective in causing their damage during S phase of the cell cycle when the new DNA is being synthesized. Endogenous (Basal mutation rate) Exogenous (Ionizing radiation, UV radiation, hydrocarbons, free radicals, chemicals)  DNA Repair Systems DNA repair is necessary not only because cells are continuously exposed to environmental mutagens but also because thousands of mutations would otherwise occur spontaneously in every cell each day during DNA replication. When defects in DNA repair mechanisms occur, mutations accumulate in the cell's DNA leading to cancer. All types of repair mechanisms are made up of enzymes that follow a general scheme of recognition, removal, repair, and relegation. However, depending on the type of damage, different enzymes are employed  Mismatch repair Corrects the mismatches of normal bases that fail to maintain normal base pairing and insertions and deletions of one or a few nucleotides that are introduced into DNA during replication. This failure is typically due to the mistakes made by DNA polymerase during replication. Recognition of a mismatch is accomplished by several different proteins including those encoded by MSH2, MLH1, MSH6, PMS1, and PMS2 genes Mutations in either of these genes predispose the person to an inherited form of colon cancer (hereditary nonpolyposis colon cancer [HNPCC]) at an early age. Base excision repair Correct the spontaneous depurination and spontaneous deamination (removal of amine groups) that happen to bases present in DNA. Spontaneous deamination of cytosine causes it to be converted to uracil. Methyl cytosine in DNA is converted to thymine on spontaneous deamination resulting in the most common mutation seen in humans a C to a T transition. Base excision repair involves recognition and removal of nucleotides that have lost the bases or have been modified Nucleotide excision repair Remove ultraviolet light–induced DNA damage as well as DNA damage from environmental chemicals. UV light can nevertheless form pyrimidine-pyrimidine dimers (thymine dimers) XERODERMA PIGMENTOSUM Autosomal recessive disorder Caused by variants in at least nine genes: DDB2, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, POLH, XPA, and XPC. These genes are involved in repairing damaged DNA (NER) The POLH gene also plays a role in protecting cells from UVR-induced DNA damage XERODERMA PIGMENTOSUM Acute sun sensitivity with marked freckle-like pigmentation of the face before age two years Sunlight-induced ocular involvement (photophobia, severe keratitis, atrophy of the skin of the lids, ocular surface neoplasms Approximately 25% of affected individuals have neurologic manifestations (acquired microcephaly, diminished or absent deep tendon stretch reflexes, progressive sensorineural hearing loss, progressive cognitive impairment, and ataxia). XERODERMA PIGMENTOSUM Hyperpigmented, freckle-like lentigines cover the entire fase and neck. The skin of the nose is atrophic and hypopigmented. AMBOSS GmbH. Xeroderma pigmentosum.https://amboss.com/. Accessed January 8th, 2025. Double-stranded DNA repair When damage from ionizing radiation, oxidative free radicals, or chemotherapeutic agents causes both the strands of DNA to be severed, two types of repair mechanisms exist to correct the damage, homologous recombination and nonhomologous end joining Homologous recombination This type of repair takes advantage of sequence information available from the unaffected homologous chromosome for proper repair of breaks. BRCA1 and BRCA2 proteins normally play a role in the homologous recombination process. Fanconi anemia is a condition caused by failures in DNA recombination repair enzymes to correct the defects by homologous recombination. Several Fanconi anemia proteins form complexes and interact with the BRCA proteins.  FANCONI ANEMIA Autosomal recessive disorder 65% patients show mutations in FANCA gene. FANCC, and FANCG genes. Accruement of chromosomal damage due to the cell's inability to conduct repairs. Cells that cannot properly repair DNA damage, resulting in genomic instability, subsequent pancytopenia, and an increased susceptibility to cytotoxic agents, UV radiation, spontaneous deformation, and predisposition to malignancies.  FANCONI ANEMIA Pre- and postnatal growth retardation Malformations of the kidneys, heart, and skeleton (absent or abnormal thumbs and radii) A typical facial appearance with small head, eyes, and mouth Cutaneous abnormalities (hyper- or hypopigmentation and cafe-au-lait spots) Bone marrow failure and susceptibility to cancer, predominantly acute myeloid leukemia.  Short thumb and café au lait spots AMBOSS GmbH. Fanconi anemia.https://amboss.com/. Accessed January 8th, 2025. Nonhomologous end joining This process permits the joining of ends even if there is no sequence similarity between them. It is error prone since it can also introduce mutations during repair. Nonhomologous end joining is especially important before the cell has replicated its DNA because there is no template available for repair by homologous recombination. Bibliography Chapter 7 Chandar, Nalini and Viselli, Susan. Lippincott's Illustrated Reviews: Cell and Molecular Biology, 2nd Edition Lippincott Williams & Wilkins, a Wolters Kluwer Health Publication, 2019. AMBOSS