SGN 19 The Molecular Basis of Inheritance

DNA directs gene expression and biological inheritance; therefore it is important to understand these processes at a molecular level Gene expression – using the gene code in DNA to make proteins and ultimately the traits of the organism Biological inheritance – the replication of DNA and the patterns of the distribution of traits to the next generation of cells and organisms

The search for the molecular basis of inheritance - what is the genetic material? Evidence (Mendel, Morgan, etc.) suggests by the early 1900’s that chromosomes contain genetic material (biomolecules responsible for bio. inh. and gene exp,) - but DNA or proteins? Many believed genetic material is proteins – heterogeneity and specificity of function vs uniformity of DNA and unapparent function

In early to mid 1900’s bacteria were critical model organisms for identifying DNA as holding genetic info

Griffith (1928) – laid the foundation for later work that would point to DNA as the genetic material 2 strains of pneumonia inducing bacteria (pathogenic/smooth and nonpathogenic/rough) Dead pathogenic mixed with living nonpathogenic induced pathogenicity in living bacteria, and their descendents Some chemical component taken up by living bacteria induced pathogenicity (changes phenotype) Transformation – change in genotype and phenotype due to assimilation of external DNA by bacteria cell

What is transforming substance? Avery (1940’s) – many purified organic substances from dead bacteria tested; only DNA induces transformation; but how does it carry genetic information?

Hershey and Chase (1952) – DNA or protein Hershey and Chase (1952) – DNA or protein? Bacteriophages transform cells into virus making factories; bacteriophage protein labeled with radioactive sulfur and found none incorporated into bacteria cells; labeled DNA with radioactive phosphorous and found it incorporated into cell

3-D structure of DNA? Watson and Crick deduce the structure of the double helix by synthesizing information collected by other scientists

Chargaff (1947) – proportion of different bases varies from species to species but ratio of guanine to cytosine always equal as is that of thymine to adenine Suggested pairing pattern of complementary bases joined with hydrogen bonds

X-ray crystallography (Franklin) provides evidence that DNA is helical and double stranded

Students should be familiar with the structure of the DNA nucleotide, the DNA single strand/molecule and the DNA double helix

Strands in double helix are antiparallel; 5’ end is anterior terminal phosphate while 3’ end is terminal posterior hydroxyl group of deoxyribose Importance of hydrogen bonds in joining 2 strands of double helix

DNA replication – occurs during the S stage of the cell cycle

2 DNA strands are complementary and, once separated, serve as templates for construction of new strands Replication is semiconservative: each new double helix is composed of one old and one new strand

Students should be able to describe the important Meselson and Stahl experiment of the late 50’s and should understand how their data supported the hypothesis that DNA replication is semi conservative

Details of replication (as understood in prokaryotes, with similarities to eukaryotes)

Begins at origin of replication where enzymes break hydrogen bonds (prokaryotic and eukaryotic processes similar in many respects – single point of origin versus multiple); replication fork proceeds in either direction; at replication fork elongation catalyzed by DNA polymerase, which adds complementary nucleotides to exposed bases one by one

Triphosphate DNA monomers lose two phosphate groups, which provide energy that drives their enzyme-mediated polymerization (bonding of phosphate group to deoxyribose to form strands)

DNA strands have antiparallel arrangement (backbones run in opposite directions); using orientation of carbons in monosaccharide, strand is said to have a 5’ (prime) and 3’ (prime) end; 5’ is exposed phosphate group of terminal nucleotide; 3’ is hydroxyl group of deoxyribose at other terminus

To begin process (at origin of replication) helicase unwinds DNA helix and separates strands; single-strand binding proteins hold strands apart; 2 replication forks grow in either direction

A complication… DNA polymerase only adds nucleotides to 3’ end of growing strand; phosphate group of joining nucleotide always added to hydroxyl group of 3’ carbon of monosaccharide of previously added nucleotide New strand is built in 5’ to 3’ direction Template is “read” in 3’ to 5’ direction

Since strand can only grow in 5’ to 3’ direction, one strand grows towards replication fork while other strand grows away; elongation on one strand (leading strand) moves towards replication fork; lagging strand must grow in direction away from replication fork

Primase (an RNA polymerase that can add to template strand without proceeding nucleotides) initiates synthesis of polynucleotide (1st step of actually making daughter strand); attaches short segment of RNA primer to DNA strand, to which DNA polymerase can add nucleotides; RNA fragment is then replaced by different polymerase

Only single primer is needed on leading strand; but lagging strand must continually lay down primer and grow backwards as new DNA is exposed at replication fork; these segments that are grown in fits are called Okasaki fragments; fragments are then joined by DNA ligase

Evidence suggests all enzymes are joined together in replicating machine that reels in double strand at one end and extrudes two double strands from other end

Base pairing is not always correct and other damage can occur that compromises copying

DNA polymerase has proofreading function; many other enzymes make repairs missed by polymerase, or damage that occurs after replication (nucleotide excision repair, for example)

Need for RNA primer to initiate elongation of strand causes problems; since polymerase can only add nucleotides at 3’ end, when terminal primers at 5’ ends in linear DNA removed, polymerase cannot fill gap; each replication therefore results in a shortening of linear chromosome

Eukaryotic DNA strands have expendable end pieces called telomeres – nongene nucleotide repetitions; with each replication these shorten; therefore in older tissue strands are shorter, which might limit lifespan of stem cells, tissue and organism

In germ-line cells (produce gametes) and cancer cells, telomerase catalyzes the lengthening of telomeres, so in gametes and zygote chromosome length restored Telomerases unusual in that they are combination of protein with short RNA strands incorporated