José A. Cardé- Serrano, PhDUniversidad Adventista de las AntillasBiol 223 - GenéticaAgosto 2011

Structural role in chromatin

Present in amounts equivalent to amounts of DNA

Major histone types: H1, H2a, H2b, H3, and H4

Basic proteins Arginine and Lysine are

Abundant Highly conserved proteins

2-nm double-stranded DNA molecule 11-nm nucleosomes 30 nm chromatin fiber Organization around a central scaffold

Constricted region of the chromosome Necessary for proper segregation of

chromosomes in mitosis and meiosis

Functions of telomeres Protect the ends of linear DNA molecules

from deoxyribonucleases Prevent fusion of chromosomes ends or

terminals Facilitate complete replication of the

ends of linear DNA moleculesMost telomeres contain repetitive

sequences and a distinct structure.

Replicación – procesos por el cual la célula genera una copia de su material genético usando como molde una previa

Iniciación extensión terminación

30,000 bpm vs 3,000 bpm1 error / billón Implicaciones en los gemelos?

Each strand serves as a template

Complementary base pairing determines the sequence of the new strand

Each strand of the parental helix is conserved in a hybrid new molecule

Problema: Pregunta? Como se replicará el DNA?

3 Hipótesis posibles

Much of what we know about DNA synthesis was deduced from in vitro studies.

Primer DNA with free 3'-OH

Template DNA to specify the sequence of the new strand

Substrates: dNTPs

Mg2+

Polymerases in E. coli DNA Replication: DNA Polymerases III and I DNA Repair: DNA Polymerases II, IV, and V

Polymerases in Eukaryotes Replication of Nuclear DNA: Polymerase

and/or Replication of Mitochondrial DNA:

Polymerase DNA Repair: Polymerases and

All of these enzymes synthesize DNA 5' to 3' and require a free 3'-OH at the end of a primer

Synthesis of the leading strand is continuous.

Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.

Ori C Burbuja de replicacion DNA A protein se pegan al repeat del core de 9 bb, se pegan entre 20

y 40 DNA A se separan las cadenas se forma la burbuja DNA B helicasa y DNA C se unen para formar el fork bidireccioinal DNA T tambien de funcion desconcocida Otras entre ellas la DNA girasa y las SSB

DNA primasa = RNA pol q hace primers (cadena leading un solo primer)

lagging primosoma = DNA primasa y DNA Helicasa DNA pol III extiende DNA topo provee los ejes de rotacion haciendo los nicks SSB mantienen DNA pol I reemplaza los primers de RNA DNA ligasa unes los fragmentos de ocka DNA girasa introdue el negative supercoil (DNA topoi)

Shorter RNA primers and Okazaki fragments

DNA replication only during S phaseMultiple origins of replicationNucleosomesTelomeres

DNA polymerase -DNA primase—initiation; priming of Okazaki fragments

DNA polymerase —processive DNA synthesis

DNA polymerase —DNA replication and repair in vivo

PCNA (proliferating cell nuclear antigen)—sliding clamp

Replication factor-C Rf-C)—loading of PCNA

Ribonuclease H1 and Ribonuclease FEN-1—removal of RNA primers

Most human somatic cells lack telomerase activity.

Shorter telomeres are associated with cellular senescence and death.

Diseases causing premature aging are associated with short telomeres.

The primary transcript is equivalent to the mRNA molecule.

The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.

The primary transcript (pre-mRNA) is a precursor to the mRNA.

The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA.

After processing, the mRNA is exported to the cytoplasm for translation by ribosomes.

Messenger RNAs (mRNAs)—intermediates that carry genetic information from DNA to the ribosomes.

Transfer RNAs (tRNAs)—adaptors between amino acids and the codons in mRNA.

Ribosomal RNAs (rRNAs)—structural and catalytic components of ribosomes.

Small nuclear RNAs (snRNAs)—structural components of spliceosomes.

Micro RNAs (miRNAs)—short single-stranded RNAs that block expression of complementary mRNAs.

Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger RNAs.

Similar to DNA Synthesis except The precursors are ribonucleoside triphosphates. Only one strand of DNA is used as a template. RNA chains can be initiated de novo (no primer required).

The RNA molecule will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA nontemplate (sense) strand.

RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5’3’ direction.

In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized in the cytoplasm.

Messenger RNA molecules function as intermediaries that carry genetic information from DNA to the ribosomes, where proteins are synthesized.

RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects.

RNA synthesis occurs within a localized region of strand separation, and only one strand of DNA functions as a template for RNA synthesis.

Transcription—the first step in gene expression—transfers the genetic information stored in DNA—genes—into messenger RNA molecules that carry the information to the ribosomes—the sites of protein synthesis—in the cytoplasm.

Tetrameric core: 2 ’Holoenzyme: 2 ’

Functions of the subunits: : assembly of the tetrameric core : ribonucleoside triphosphate binding

site ’: DNA template binding region : initiation of transcription

1. Binding of RNA polymerase holoenzyme to a promoter region in DNA

2. Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template

3. Formation of phosphodiester bonds between the first few ribonucleotides in the anscent RNA chain

The transcript initiation site is +1.

Bases preceding the initiation site are given minus (–) prefixes and are referred to as upstream sequences.

Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences.

Consensus sequences: -10 sequence and -35 sequence

Recognition sequence: -35 sequence

Rho-dependent terminators—require a protein factor ()

Rho-independent terminators—do not require

RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3) termination.

RNA polymerases—the enzymes that catalyze transcription—are complex multimeric proteins.

The covalent extension of RNA chains occurs within locally unwound segments of DNA.

Chain elongation stops when RNA polymerase encounters a transcription-termination signal.

Transcription, translocation, and degradation of mRNA molecules often occur simultaneously in prokaryotes.

Three different enzymes catalyze transcription in eukaryotes, and the resulting RNA transcripts undergo three important modifications, including the excision of noncoding sequences called introns. The nucleotide sequenced of some RNA transcripts are modified posttranscriptionally by RNA editing.

A 7-Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage.

A poly(A) tail (a 20-200 nucleotide polyadenosine tract) is added to the 3’ end of the transcript. The 3’ end is generated by cleavage rather than by termination.

When present, intron sequences are spliced out of the transcript.

TFIID se une a TATA boxTFIID – contiene TBP

TFIIA se une al complejo

TfIIB

Dnase Fingerprinting

TFIIF – helicasa - iniciacion- se asocia primero a la pol y luego ambos al complejo

Polimerasa II

TFIIE

TFIIH y TFIIJ-ubicacion desconocidaH – helicasa para extension

-1ra modificacion del preMRNA

-adicion de guanosinsa metilada

-enlace 5’-5’

-se anade cotranscripcional

-2 funciones- iniciacion de traduccion- estabilidad/proteccion

Terminación - -por corte endonucleolitico del pre mRNA-varios puntos, 1000-2000 down de donde sera el 3’-AAUAA y GU- poliA pol añade 200 A-2 funciones:

-estabilidad-transporte

Usually the genetic information is not altered in the mRNA intermediary.

Sometimes RNA editing changes the information content of genes by Changing the structures of individual

bases Inserting or deleting uridine

monophosphate residues.

Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm.

Introns (or intervening sequences) are noncoding sequences located between coding sequences.

Introns are removed from the pre-mRNA and are not present in the mRNA.

Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mRNA after splicing.

Introns are variable in size and may be very large.

Removal of introns must be very precise.

Conserved sequences for removal of the introns of nuclear mRNA genes are minimal. Dinucleotide sequences at the 5’ and 3’

ends of introns. exon-GT…………AG-exon TACTAAC box about 30 nucleotides

upstream from the 3’ splice site.

RNA/protein structure Excises introns from

nuclear pre-mRNA

Five snRNAs: U1, U2, U4, U5, and U6

Some snRNAs associate with proteins to form snRNAs (small nuclear ribonucleoproteins)

2 pasos-corte enel lado 5’ del intron-formacion de enlace intramolecular entre G5’ y A2’ al lado 3’-requiere spliceosome completo y ATP-formacion del lazo-corte del lado 3’ y union de los dos exones 5’3’

Mutation A change in the genetic material The process by which the change occurs

Mutant—an organism that exhibits a novel phenotype

Types of Mutations Changes in chromosome number and

structure Point mutations—changes at specific sites in

a gene (substitution, insertion, or deletion)

Germinal mutations occur in germ-line cells and will be transmitted through the gametes to the progeny.

Somatic mutations occur in somatic cells; the mutant phenotype will occur only in the descendants of that cell and will not be transmitted to the progeny.

Spontaneous mutations occur without a known cause due to inherent metabolic errors or unknown agents in the environment.

Induced mutations result from exposure or organisms to mutagens, physical and chemical agents that cause changes in DNA, such as ionizing irradiation, ultraviolet light, or certain chemicals.

Forward mutation—mutation of a wild-type allele to a mutant allele.

Reverse mutation (reversion)—a second mutation that restores the original phenotype. Back mutation—a second mutation at

the same site. Suppressor mutation—a second

mutation at a different location in the genome.

Isoalleles have no effect on phenotype or small effects that can be recognized only by special techniques.

Null alleles result in no gene product or totally nonfunctional gene products.

Recessive lethal mutations affect genes required for growth of the organisms and are lethal in the homozygous state.

Mutations may be dominant or recessive.

Because of the degeneracy and order in the genetic code, man mutations have no effect on the phenotype of the organism. These are called neutral mutations.

Adult hemoglobin (Hemoglobin A) contains two chains and two chains.

Hemoglobin in patients with sickle-cell anemia (Hemoglobin S) differs from Hemoglobin A at only one position.

The sixth amino acid in the chain is glutamic acid in Hemoglobin A and is valine in Hemoglobin S. This substitution is caused by mutation of a single base pair.

Tay-Sachs disease is an autosomal recessive disease.

The mutation causing Tay-Sachs disease is in the gene encoding hexosaminidase A.

Phenylketonuria is caused by a mutation in phenylalanine hydroxylase.

Albinism is caused by a mutation in tyrosinase

Alkaptonuria is caused by a mutation homogentisic acid oxidase.

Tyrosinosis is caused by a mutation in tyrosine transaminase.

Tyrosinemia is caused by a mutation in p-hydroxyphenylpyruvic acid oxidase.

A transition replaces a pyrimidine with another pyrimidine or a purine for another purine.

A transversion replaces a pyrimidine with a purine or a purine with a pyrimidine.

Accuracy of the DNA replication machinery

Efficiency of the mechanisms for the repair of damaged DNA

Degree of exposure to mutagenic agents in the environment

Induced mutations occur upon exposure to physical or chemical mutagens.

Hermann J. Muller and Edgar Alternburg measured the frequency of X-linked recessive lethal mutations in Drosophila.

Muller demonstrated that exposing Drosophila sperm to X-rays increased the mutation frequency.

Hydrolysis of cytosine to a hydrate may cause mispairing during replication

Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate error-prone DNA repair mechanisms.

The Ames test provides a simple and inexpensive method for detecting the mutagenicity of chemicals.

Bruce Ames and coworkers developed an inexpensive and sensitive method for testing the mutagenicity of chemicals with histidine auxotrophic mutants of Salmonella.

A DNA repair endonuclease or endonuclease-containing complex recognizes, binds to, and excised the damaged base or bases.

A DNA Polymerase fills in the gap, using the undamaged complementary strand of DNA as a template.

DNA ligase seals the break left by DNA polymerase.

Base excision repair pathways remove abnormal or chemically modified bases.

Nucleotide excision repair pathways remove larger defects, such as thymine dimers.

Individuals with XP are sensitive to sunlight.

The cells of individuals with XP are deficient in the repair of UV-induced damage to DNA.

Individuals with XP may develop skin cancer or neurological abnormalities.

The importance of DNA repair pathways is documented convincingly by inherited human disorders that result from defects in DNA repair.

Certain types of cancer are also associated with defects in DNA repair pathways.