DNA Replication and Repair Mechanisms

• DNA replication needs DNA polymerase, subunits, and accessory factors to replicate the strand
• DNA polymerase reads the template in the 3' to 5' direction
• DNA polymerase adds to the template in the 5' to 3' direction
• Nucleases need a 3' hydroxy group to add new nucleotides to the strand
• In order to replicate new DNA, a template is necessary, along with dNTPs, enzyme, and a primer

Replication of DNA

• Eukaryotes use DNA polymerases 1 and 3 in DNA replication
• DNA polymerase reads the template in the 3' to 5' direction
• DNA polymerase adds the template in the 5' to 3' direction
• DNA polymerase families include A, B, C, D, X and Y

End Replication Problem

• Priming is necessary for DNA polymerase and thus usually involves RNA, which is later degraded, resulting in a region of ssDNA that can't be replicated
• ssDNA degrades via DNA repair machinery, leading to shorter chromosomes after each replication round
• Around 30 to 50 replication cycles, the cell can undergo chromosome shortening, causing cells to undergo replicative senescence and cell cycle arrest
• Telomeres are there to solve the problems of end replication
• RNA primers need to be removed, which leaves a gap at the end of chromosome
• The above isn't an issue with circular DNA
• The ssDNA gap causes cells to respond
• DNA replication takes place on 2 strands, which each have different polarity
• Polarity introduces end replication problem

Telomeres

• Telomeres serve two functions including protecting the ends of a chromosome and provide a way of overcoming the end-replication problem
• Telomeres can shorten normally with aging, dysfunction contributes to age-related disease, genome instability, and cancer
• Human telomeres consist of many repeats of the sequence 5' TTAGGG 3'
• Consists of formation, function to protect the chromosomes end, telomeric shortening contributes to ageing, and consists of 5' TTAAGGG 3'
• G-rich strand extends 5' to 3' towards a chromosome end and terminates in a ss region called the G-rich overhand
• Repetitive sequence varies in length in species, from 50 bp to 30,000 bp (30 kb)
• G-rich srand: strand that extends 5' to 3' towards a chromosome's end and terminates in a small single stranded region named the G-rich overhang
• Length of telomere sequences on chromosomes differ

Telomeres Protection

• G-rich overhang is processed into a t-loop, through base pairing between the ss G-overhang and the ds region of telomere DNA
• Telomere t-loop configuration helps prevent the inappropriate activation of the cell's DNA damage response (nuclease activity and cell death)
• telomeres form loop structures such as a T-loop, and a D-loop
• Cells do not like having single stranded DNA
• Telomeres, forms the t-loop structures to compensate; structure contains repetitive G-rich overhangs that loop over
• D-loop is a displacement loop, or when non-sisters exchange genetic material

Telomere Binding

• Telomere-binding proteins are for telomere function and are known as shelterin proteins
• Shelterin's 3 classes: end-binding proteins, dsDNA-binding proteins, and telomere-associated proteins that don't bind DNA.
• There's also POT1, TIN2, TPP1, RAP1, TRF2 TRF1
• POT1 binds ss G-overhangs and associates with TPP1
• TRF1 + TRF2: binds along length of double-stranded telomeric DNA and interacts with TIN2
• The t-loop is stabilized by the proteins, which protect the ends, and they regulate their length by controlling the access of its telomerase
• The regions for protein binding is the shelterin complex and includes those that bind to the end, those that bind to the double stranded DNA itself, and telomere-associated proteins.

Telomerase

• Telomere length is maintained by the enzyme telomerase
• Telomerase: large ribonucleoprotein (RNP) consists of telomerase reverse transcriptase (TERT) enzyme & non-coding templating RNA (TERC, humans)
• Telomerase uses 3'-OH of G-overhang to bind complementary RNA that acts as temp for new DNA, it then adds a sequence to the G-overhang
• Opposite strand is filled in via DNA polymerase
• Two parts that are important to telomerase activity are TERT ,telomerase reverse transcriptase and TERC, non-coding template
• Telomerase adds a 6-nucleotide sequence to the end of the chromosomes
• Cells that aren't continually dividing stabilize their tips with t-loops
• One aspect includes a gap that can't be filled by nucleotides
• Telomerase extends the G-overhang, thus the other end can be filled in

Telomere Maintenence

• Telomerase activity is regulated tightly and is highly expressed in stem cells and germ line
• Telomeres shrink with age, especially in rapidly cycling cells like hematopoietic, and epidermal stem cells
• Senescence or cell death be triggered w as little as 5 of 46 telomeres that are super short
• Mutation in genes that encode the RNA or telomere proteins can lead to dyskeratosis, aplastic anemia, idiopathic pulmonary fibrosis.
• Multi-generational consequences can happen if the germ lines telomeres affected
• Mutation in a gene regulates the shelterin complex is important for recruiting tips for extending telomerase

DNA Damage

• DNA Damage is either repaired or becomes a mutation during replication
• Scale and type can create point mutations which affect only a few nuceleotides or chromosome or karyotype altering which changes a gene number
• Source can stem from normal error in normal process, erros caused by defective process, or environmental mutagens such as chemical, UV, radiation and heavy metals
• Phenotypic consequence can be thought of by the scale and source of damage, Point Mutations are smaller w proofreading activity and need considering of whether the non coding region

Point Mutation

• Point mutations can be substitution, deletions, or insertion
• Substitution: Purine to pyrimidine is a transversion whereas A to G or C to T is a transition, which is purine to purine or pyrimidine to pyrimidine
• C-T transitions happen twice as often in humans than all other mutations together

Single Nucleotide Lesions

• Dividing cells may be subject to ~70,000 instances of DNA damages daily
• Most of these get repaired by BER pathway
• Type of lesions are Hydrolysis, Methylation, Oxidation w 9,000 to 10,000 being the common issue
• Damage can be oxidation, hydrolysis, and methylation which change their substituents

DNA Repair

• Alterations that require repair can involve oxidation, methylation, or hydrolysis
• Some metabolism byproducts may come from reactive oxygen species
• In Deupruination, the nucleotide loses its nitrogenous base and in deamination a amino group gets removed leading to changes
• DNA also gets damages in sugar structure or with Thymidine Dimers as well Oxidation damage
• Oxidative damage: 8-oxodG mispairs with A Normal G-C → mutant T-A after replication
• ds breaks are the most dangerous, caused by ionization radiation so the mechan is to involve homologous recombination for the repair.
• UV light bad for DNA by making dimers instead of watson crick

Normal Error Process

• DNA polymerase has high fidelity, making few errors in nucleotide matching
• DNA polymerase has a proofreading mechanism, where it will stop if the wrong nucleotide is inserted
• The 3' to 5' exonuclease recognizes and excises mismatches
• Eukaryotic DNA polymerases make errors that can range from 10^4 to 10^6
• Exonuclease proofreading decreases the chance of making mistakes w low count in bacteria

Errors and Defects

• Errors cause mutations and defects when cancerous
• Mismatch repari involves the MutSa-MutLa pathway, to help repair replication erros as a vast majority
• Mismatch repair pathway: in cells that are active & dividing with a main goal of fixing majority of error
• Recruits accessory proteins: cut strand and mismatches where new repalce from DNA as well nick sealed by DNA ligase
• PCNA the cap based on the location problem distinguished to

The Repair and Damage

• nucleotide mismatches, double-strand breaks (DSBs), base damage , sugar damage, DNA cross-linking are all types od DNA damage

Base Excision Repair

• Repairs include DNA glycosylases (11 in mammals), recognize a specific type of altered base in DNA and catalyze its hydrolytic removal
• Endonuclease cuts phosphodiester backbone such as where the resulting gap is repaired can be found for differentiated cells and monitor through monitoring as Rracil because it's been deaminated and recruit is the DNA in it. Exonuclease cuts the nucleotides, DNA Ligase Repairs the strand.

Nucleotide Exision Repair

• repairs the damage caused by almost any large change in the structure of the DNA double helix
• Process Involves XPC/RAD23B recognition, Nick recognition, TFIIH strand opening with exonucleases filling and ligating up via ployremase.

Stop Signal

• Mutating and division causes a cell's DNA damage causing a signaling pathway

The Kinase

• kinases can be ATM or ATR, which recruit Chk1 protein kinases, the phosphoralation of the p53 which normally is in ubiquitylation and destruction
• Important not to pass to the next division
• P53 a tumer suppressor, tells the cells where stop / repair normally low cells as the Mdm2 keeps is going, which inhibits the Cyclin which halts everything.
• ATR and ATM kinases reactivate in response to DNA damage and phosphoralate P53
• ATR / ATM kinases that can either single or or double the strand and bind to many gens

Errors Caused by Defect

• Mutate from replication can lead to various diseases that causes issues for human

Trinucleotide repeater

• Leads to Huntington Disease which leads neuro issues and survival issues later on with expanded of glutamine toxicity
• Polymerase slips
• Defect leads to expansion on the chromosome to continue for Huntington

Strands

• Cross links forms between DNA strands which with blocks replications and transcript

Double Strand

double-strand breaks(DSBs)

• held together by hydrogren bnds and covalently with scale
• Aneuploidy, less or more chromosomes than normal from meiosis

Karyotypes Reveal Issues

• Karyotype reveals Chromosomal abnormalities such as translocations, inversions, and aneuploidys such as Down’s syndrome, Turner’s syndrome, and Klinefelter’s syndrome.

Cancer

• cancer from chromosome and abnormality:duplication,deletion or rearrangements with heterogeneity

Double Stand Breaks

• Can cause repairs by
• HDR Homologous directed repair AKA homologous recombination that has errors and has a template. Uses identical sister chromosomes that can cause similar homolouges.
• NHEJ Non-Homologous End Join - end of life coming and fusings causing nucleotides badness that mutates.

Somatic Translocations

• human cancers such philidelphi Chromosome can cause repairs of break leads to fusion of new genes

Explore the intricate processes of DNA replication, telomere maintenance, and DNA repair mechanisms. Uncover the roles of key enzymes and proteins involved in maintaining genomic stability. Questions cover from dealing with single-stranded DNA breaks to cell cycle arrest.