DNA Replication: Key Concepts

• DNA replication ensures genetic information is accurately passed to daughter cells during cell division.

Key Terms in DNA Replication

• Antiparallel: DNA strands run in opposite directions (5' to 3' and 3' to 5'), crucial for replication and base pairing.
• Double Stranded: DNA is typically composed of two strands that form a double helix structure
• Single Stranded: Structure with only one nucleotide strand, common during replication or in RNA.
• 5’ end: The end of a nucleotide chain where the 5th carbon of the sugar is free
• 3’ end: The end of a nucleotide chain where the 3rd carbon of the sugar has a free hydroxyl
• Complementary: Adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G) in DNA; uracil (U) replaces thymine in RNA..
• Base Pairing: Hydrogen bonds between nucleotides ensure accurate replication and transcription (A-T, G-C).
• Template strand: Original DNA strand used to synthesize a new strand
• Daughter strand: The newly synthesized strand that is complementary to template strand
• Polarity: Refers to the 5' to 3' directionality of nucleic acid strands, important for enzymatic processes.
• Origin of replication: Specific DNA sequence where replication starts.
• Leading strand: Synthesized continuously towards the replication fork.
• Lagging strand: Synthesized discontinuously away from the fork in Okazaki fragments.
• Primer: Short RNA segment synthesized by primase, providing a starting point for DNA polymerase.
• DNA polymerase: Synthesizes new DNA by adding nucleotides to the 3' end.
• Primase: Synthesizes the RNA primer needed to start DNA synthesis.
• Ligase: Seals nicks in the DNA backbone by joining Okazaki fragments.
• Helicase: Unwinds and separates double-stranded DNA into single strands.
• Single strand binding protein: Prevents single-stranded DNA from re-annealing or degrading.
• Telomere: Repetitive sequences at the ends of eukaryotic chromosomes, preventing degradation and fusion.
• Telomerase: Extends telomeres, counteracting shortening in germ, stem, and cancer cells.

What Must Genetic Material Be Able to Do?

• Replicate accurately.
• Store information effectively.
• Be transmitted from parent to offspring reliably.
• Allow for variations among individuals and species.

Griffith and Avery Experiments

• Disproved proteins as genetic material, showing DNA can transform cell properties.
• R (non-virulent) and S (virulent) bacterial strains differ in appearance and disease-causing ability.
• Heat-killed S strain mixed with live R strain caused death in mice, inheriting the ability to kill.
• R bacteria were transformed into virulent S bacteria by a heritable substance.

Transforming Principle

• A heritable component transforms non-virulent R strain bacteria into virulent S strain.

Identifying the Transforming Substance

• Soluble extract from heat-killed S bacteria treated with enzymes destroying protein, DNA, lipid, or polysaccharide.
• Bacterial transformation lost when DNA is destroyed by nucleases.
• DNA, not protein, is responsible for heritable changes during bacterial transformation.

Base Compositional Analysis (Chargaff's Rules)

• Amount of adenine (A) equals thymine (T), and guanine (G) equals cytosine (C) in DNA (A=T, C=G).
• Suggests base pairing.

X-Ray Diffraction

• Rosalind Franklin and Maurice Wilkins showed DNA has a helical structure with a uniform diameter.

Data Modeling (Watson and Crick)

• Deduced DNA's double helix structure by modeling diffraction data, dNTP structure, and Chargaff's rules.
• Hydrogen bonds between A-T (two bonds) and G-C (three bonds) allow purine/pyrimidine fit.
• Complementarity allows predicting the opposite strand sequence.

DNA Structure

• Antiparallel double helix formed by complementary base pairing of nucleotides.
• Conforms to Chargaff's rules.
• Purine pairing with pyrimidine ensures a uniform diameter.

Nucleotides

• Nucleotides are the building blocks of nucleic acids.
• Nucleotides covalently connected by phosphodiester bonds between 3'OH and 5' phosphate.
• Phosphate connecting sugars contributes negative charge to the sugar-phosphate backbone.
• DNA strand has directionality (5' and 3' ends).
• Strand contains a specific sequence (e.g., 5'-TACG-3').

DNA Replication Overview

• Parent molecule has two complementary strands (A with T, G with C).
• DNA strands separate.
• Each parental strand serves as a template to determine the order of nucleotides along a new strand.
• Nucleotides connect to form sugar-phosphate backbones of new strands.
• Each daughter DNA has one parental and one new strand.
• DNA polymerase catalyzes DNA synthesis in the 5' to 3' direction.
• Incoming dNTP base pairs with the template.
• Phosphodiester bond forms between the 3' OH of the terminal nucleotide and 5' phosphate of incoming dNTP.
• Energy comes from hydrolysis of 2POr-, coupled to polymerization.
• Reaction creates a new 3'OH for the next nucleotide.
• DNA synthesis requires a primer; DNA polymerase adds nucleotides to the 3' end of a pre-existing strand.
• Primase produces a short, complementary RNA primer.

DNA Polymerases

• E. coli has 5 DNA polymerases (I and III are major).
• Humans have 15 DNA polymerases.

Origins of Replication

• Replication begins at specific origin sites where DNA strands separate.

Origins of Replication in Prokaryotes

• E. coli's circular DNA has one origin.
• Replication proceeds bidirectionally until the entire molecule is copied.

Origins of Replication in Eukaryotes

• Chromosomes are linear and long.
• Have hundreds or thousands of origins per cell (up to 80,000 in humans).
• Multiple origins allow faster DNA replication.

Enzymes in DNA Replication

• DNA helicase: Unwinds double helix by breaking hydrogen bonds.
• DNA polymerase: Catalyzes phosphodiester bond formation, extending the new DNA strand.
• Primase: Synthesizes RNA primer using parental DNA as a template.
• Single-strand DNA binding protein: Prevents separated strands from rejoining.
• Topoisomerase: Relieves torsional strain ahead of the replication fork.

Leading and Lagging Strands

• DNA polymerase works 5' to 3'.
• Leading strand: DNA polymerase moves continuously towards the fork.
• Lagging strand: Made of Okazaki fragments, joined by DNA ligase.
• Lagging strand synthesis is away from the replication fork.

Bidirectional Replication

• Separation at a single origin leads to two forks moving in opposite directions.
• Leading strand requires one primer; lagging strand requires multiple primers.
• DNA polymerase has 3' to 5' exonuclease proofreading activity.

The End Replication Problem

• After the last RNA primer is removed from the 5' end of the lagging strand, a shortened 5' end remains.
• Each replication round causes chromosome shortening.
• Bacteria have circular chromosomes, avoiding this problem.
• Eukaryotes use telomeres to protect chromosome ends.

Telomeres

• Specialized DNA sequences at the ends of eukaryotic chromosomes.
• In humans, many repeats of 5'-GGGTTA-3'.
• Telomere sequence length varies among species (up to 10,000 nucleotides).
• Telomerase enzyme repairs DNA ends in some cells.
• Telomere length is linked to repeated DNA replication andAging.
• Telomerase is active in embryonic and highly proliferative cells.
• High telomerase activity is in 90% of human cancers.

Cell Division

• Reproduction in single-celled organisms.
• Growth and development in multicellular organisms.
• Cell replacement and tissue renewal.

Asexual Reproduction

• Asexual cell division results in genetically identical daughter cells.
• DNA is precisely replicated and passed equally to daughter cells.
• DNA replication and cell division timing are coordinated.

Cellular Organization of Genetic Material

• All DNA in a cell constitutes the genome.
• Genome can be a single DNA molecule (prokaryotes) or multiple (eukaryotes).
• DNA molecules are packaged into chromosomes.
• Eukaryotic chromosomes: Linear DNA with 100s-1000s of genes, associated with proteins (chromatin).
• Every species has a characteristic number of chromosomes.
• Human somatic cells have two sets of chromosomes (23 pairs); gametes have half as many (23).

Functional Chromosome Requirements

• Must have three DNA sequences.

Cell Division Processes

• Doubling the genome (chromosome replication).
• Separating the duplicated genome (chromosome segregation).

Bacterial Cell Division (Binary Fission)

• Bacteria have a circular chromosome attached to the plasma membrane.
• Each new DNA molecule remains attached to the PM.
• Cell elongates, and the plasma membrane grows inward, pinching off two cells.

Eukaryotic Cell Division

• Eukaryotes have more than one chromosome.
• Mechanisms regulate chromosome replication, separation, and partitioning.
• Chromosomes replicate during the S phase, producing sister chromatids connected by cohesin proteins until separated
• Separate the two duplicated chromosomes
• Distribute chromosomes equally between daughter cells
• Mitosis, a part of the cell cycle, accomplishes steps 2-4.

Cell Cycle

• Sequence of events in the life of a dividing eukaryotic cell.
• Cells divide at a specific time, highly regulated.

Phases of the Cell Cycle

• M (Mitosis): Cell division.
• G1 (First Growth): Cell growth.
• S (Synthesis): DNA replication.
• G2 (Second Growth): Further cell growth.
• Interphase: When cells aren't dividing.

Mitotic (M) Phase

• Mitosis: Separation of replicated chromosomes/nuclear division.
• Cytokinesis: Cytoplasmic division, forming two daughter cells.

Chromosome Structure During Cell Division

• Before division, DNA is replicated into sister chromatids held together by cohesin.
• The centromere is the constricted DNA sequence.
• During mitosis, sister chromatids separate and move to opposite ends.
• Separated chromatids are called chromosomes.

Interphase Events

• Chromosomes replicate in the S phase.
• Microtubule organizing centers (MTOCs) replicate in late interphase.
• Animal cells use centrosomes for mitotic spindle formation.

Centrosomes

• Duplicate during interphase.
• Separate and move to opposite poles during prophase.

Kinetochores

• Protein complexes on centromere DNA of sister chromatids.
• Microtubules attach to chromosomes via kinetochores.
• Defects in kinetochore function lead to chromosome instability and cancer.

Mitotic Spindle

• Microtubule-based machine using polymerization/depolymerization of tubulin.
• Provides the force for chromosome alignment and sister chromatid separation.

Microtubule Types

• Astral: interacts with cell membrane.
• Kinetochore: attach to kinetochores on chromosome
• Overlap: interact with each other
• Kinetochores face opposite directions, pulling sister chromatids apart during anaphase.

Mitosis Phases

• Mitosis = nuclear division
• cytokinesis= cytoplasmic division
• Prophase: Chromosomes condense; mitotic spindle forms, pushing centrosomes apart.
• Prometaphase: Nuclear membrane fragments; microtubules attach to chromosomes via kinetochores.
• Metaphase: Chromosomes align midway between poles at the metaphase plate due to equal and opposite forces.
• Anaphase: Sister chromatids separate, pulled by spindle fibers.

Anaphase Molecular Mechanism

• Cohesins hold sister chromatids together.
• Anaphase-promoting complex (APC) cleaves cohesins, allowing sister chromatids to separate.

Telophase

• Nuclear membrane reassembles.
• Chromatin becomes less tightly coiled.
• Cytokinesis: cytoplasmic division These processes start with one cell and produce two genetically identical cells

Mendel's Experiments

• Parents (P) (pure-breeding) purple flowers bred with white flowers yielded all purple flowers (F1 generation).
• F1 offspring resembled only one parent, refuting the blended inheritance theory.

Dominance and Gene Dosage

• In many cases dominance may simply be a function of gene dosage Tay-Sachs is a fatal neurodegenerative disease. TS is inherited in an autosomal recessive pattern. Caused by a recessive lethal mutation that affects an enzyme that breaks down lipids in nerve cells. Accumulation of lipids in brain causes nerve defects and premature death (children live ->3-6 yr). Tay-Sachs heterozygote has normal phenotype but produces half the amount of hexosaminidase as the homozygous wild type individual. TS heterozygote appears completely dominant at the phenotypic level but incompletely dominant at the biochemical level.

Incomplete dominance in carnation flower color

This intermediate phenotype appears to support ”the blending hypothesis” but note that the traits of the parental generation re-appear in their original forms in the F2 generation. Inheritance is “particulate”, as predicted by Mendel. RR=Red, rr=white, but heterozygote offspring have intermediate phenotype (pink).

Co-dominance-

• Each of the two alleles at a given locus have phenotypes in the heterozygote
• Example of co-dominance:: The ABO blood group antigens in humans regulated by I gene that encodes glycosyltransferase. The different alleles affect the substrate specificity of this glycosyltransferase Four blood types are produced by different combinations of multiple (3) alleles Although three I alleles exist in population, any individual has only two. Co-dominance and incomplete dominance can be distinguished from one another by the phenotype of the heterozygote. Incomplete dominance = intermediate phenotype Co-dominance = The phenotype of both alleles is expressed

Multiple Alleles

• Within a population, there may be many different alleles of a particular gene
• Example: Coat color in rabbits is determined by one gene with four alleles: C > cch > ch > c C determines dark gray cch determines chinchilla ch determines light gray c determines albino Different combinations of the coat color alleles show a hierarchy of dominance. Remember: Any one individual has only two alleles of a particular gene, one from each parent

Misconceptions about Dominant and Recessive Alleles

• commonly assumed that dominant allele is the “normal, wild type”, and the recessive is the mutant,
• also assumed that dominant allele is the one found most frequently in a population.
• However dominant alleles are not necessarily found more frequently among the population nor are they always the “wild type”.
• Pp (or PP) polydactyly cat and human
• Example: Polydactyly allele (P) leads to growth of extra fingers or toes; is autosomal dominant mutation. Found in population (~1:1000). Most cats (and people) with normal number of digits are recessive (pp) homozygotes. Because P is dominant, heterozygous parent with trait has 50% chance of transmission to offspring. (Pp X pp) = Pp+ Pp+ pp +pp = 1/2 Pp (mutant) +1/2 pp (wild type)

Homozygous Lethal Dominant Alleles

• Can cause altered phenotypic ratio where 2:1 ratio of manx to non-manx kittens because MM are embryonic lethal Short or tail-less Manx cat is due to dominant allele

Complex Inheritance Patterns

• Factors that complicate simple Mendelian inheritance patterns include
• 1.Incomplete Dominance -the phenotype heterozygous is intermediate between those of the two homozygotes.
• 2.Codominance-the phenotype of heterozygote has some aspect of both alleles
• 3. Multiple alleles - two or more alleles at a locus produce two different phenotypes that both appear in heterozygote (one allele does not ”overpower” the other).
• 4. Epistasis - A gene at one locus affects the phenotypic expression of a gene at a second locus.
• 5. Pleiotropy - the ability of a gene to affect an organism’s phenotype in many different ways. Environment - can have a profound effect on phenotype.

X Chromosome Inactivation

• Females inherit two X chromosomes, but only one X chromosome is active in most cells. The X chromosome During female development, one X chromosome per cell condenses into a compact Barr body. This condensation inactivates most of X genes so both males and females have same dose (one copy) of genes on the X chromosome. The condensed Barr body chromosome is reactivated in ovarian cells that produce eggs, so every female gamete has an active X.
• The choice of which X chromosome will be inactivated is random. After Barr body formation, all mitotic descendents have the same inactive X. Therefore, females are a mosaic of cells, ~50% with active paternal X, 50% with active maternal X. Selection of X chromosome inactivation occurs randomly in embryonic cells... Women are mosaics for X chromosomes

Mosaic Inheritance

• The orange and black pattern on tortoise shell/calico cats is an example of this mosaic inheritance and is due to patches of cells expressing the X-linked orange or the black fur allele.

Sex-Linked Genes

• In addition to their role in determining sex, the sex chromosomes, especially the X chromosome, carry genes for many characters unrelated to sex. Sex-linked genes have unique patterns of inheritance Transmission of X-linked recessive traits. Below, a recessive X-linked mutant allele is denoted by a, and wild type is A. White boxes are unaffected progeny, light pink are heterozygous “carriers” , and dark pink are homozygous mutant progeny Mutant father transmits the mutant allele to all daughters but no sons. Mother is homozygous WT so all daughters are heterozygous but none are affected. Female heterozygote who mates with normal male transmits mutant allele to ½ daughters and ½ sons. 50% chance that each daughter will be carrier. 50% chance that each son will have disorder. A female carrier who mates with an affected male. There is a 50% chance of each child being affected, no matter what sex. Daughters who appear normal are all heterozygous. Homozygous recessive female offspring requires heterozygous mother AND affected father There are ~ 1000 genes on the X-chromosome.

Examples of X-Linked Recessive Conditions

• These include: red/green color-blindness, male pattern baldness, hemophilia, and Duchenne muscular dystrophy
• These mutations are rare, recessive, and X- linked. Therefore, far more common in males than females. Chance of female inheriting double dose of mutant allele is much less than male inheriting single dose. A classic example is male pattern baldness

Sex Chromosome Aneuploidies

• • Unlike autosomes, aneuploidies of sex chromosomes are relatively well tolerated. The Y chromosome carries few genes, none of which are essential. Extra X chromosomes are inactivated as Barr bodies. In cells with >1 X chromosome, all but one are inactivated. X Y XX O XXX XXY XO OY Triple X Klinefelter Turner Inviable