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The Chemical Structure, Replication, and Manipulation of DNA

Genome Size

• Complex organisms have large genomes=genetic contents of a cell

• Genomic size increases with evolutionary complexity

• Size of DNA is measured in kb=kilobase pairs

• Size of large genomes is measured in Mb=megabase pairs

DNA: Chemical Composition

• Gene = unique sequence of DNA bases

• Two types of nitrogen-containing bases comprise the chemical structure of DNA:

- purines = adenine and guanine

- pyrimidines = thymine and cytosine

DNA: Chemical Composition• Hydrogen bonds between purines and pyrimidines

form the double-strand structure of DNA • Nucleotides = building blocks of DNA = phosphate

+ sugar + base• Nucleoside = sugar +base• Sugar = 5 carbon

deoxyribose• Phosphodiester bonds

link sugar molecules to phosphate groups

DNA: Chemical Structure

• DNA nucleotides = a chain of bases • Orientation of sugar-phosphate linkages = 5’

to 3’ as the phosphate attached to the 5’ carbon of one sugar is linked to the 3’ carbon of the next sugar

• Purine and pyrimidine bases are linked to the 1’ carbon of sugar

DNA: Chemical Structure

• DNA consists of two polynucleotide chains which run 5’ to 3’ in opposite directions = antiparallel

• DNA chains are held together by hydrogen bonds between bases

• DNA bases pair by Chargaff’s rules:

- Adenine (A) pairs with Thymine (T)

- Guanine (G) pairs with Cytosine (C)

DNA: Watson-Crick Model

3-D structure of the DNA molecule:• DNA is a double helix consisting of two

polynucleotide chains held together by hydrogen bonds between the purines and pyrimidines

• Helix forms major and minor groove

• Diameter of the helix = 20 Angstroms

• Each turn of the helix = 10 bases = 34 Angstroms

DNA Replication

Watson-Crick model of DNA replication

• Hydrogen bonds between DNA bases break to allow strand separation

• Each DNA strand is a template for the synthesis of a new strand

• Template (parental) strand determines the sequence of bases in the new strand (daughter)= complementary base pairing rules

DNA Replication

• Semi-conservative = each original DNA strand is a template for a new strand complementary to the original

• Meselson and Stahl showed that newly synthesized DNA consists of one original strand (unlabeled) and one new strand labeled during synthesis with a heavy isotope of nitrogen

DNA Replication

• Autoradiogram of replicating circular chromosome shows that DNA synthesis is bi-directional from a single start site = origin of replication (OR)

• replication forks = region where parental strands are separating and new strands are synthesized

Circular DNA Replication

• Movement of the replication fork is aided by topoisomerases = enzymes which unwind the DNA helix to permit strand separation

• DNA topoisomerase I unwinds DNA by cutting one strand, rotating it around the second strand and then sealing the single strand break (nick)

Linear DNA Replication

• Replication of linear DNA molecules proceeds bidirectionally from multiple origins of replication which form replication loops

• Replication continues to expand the replication loops until they fuse to form two separate molecules of DNA

• The replicated DNAs contain the same DNA sequence

Rolling Circle Replication

• One DNA strand is cut by a nuclease to produce a 3’-OH extended by DNA polymerase

• The newly replicated strand is displaced from the template strand as DNA synthesis continues

• Displaced strand is template for complementary DNA strand

DNA Synthesis

• Addition of nucleo-tides into growing DNA chain by DNA polymerase occurs by cleavage of two phosphate groups and the attachment of the nucleoside monophosphate to the 3’-OH of adjacent deoxyribose sugar

• Complementary base pairing with template specifies new strand order

DNA Synthesis

• DNA polymerase extends a chain of nucleotides in 5’- to- 3’ direction only

• Template strand is antiparallel

• DNA polymerase proofreading function = 3’-to-5’ exonuclease which removes mismatched bases

DNA Synthesis

Each replication fork consists of:• Leading strand: continuous synthesis• Lagging strand: discontinuous synthesis• DNA polymerase

synthesizes lagging strand in short segments = Okazaki fragments

DNA vs. RNA

• DNA sugar = deoxyribose• RNA sugar = ribose• RNA contains the pyrimidine uracil (U) in

place of thymine (T)• DNA is double-stranded• RNA is single-strand• RNA = primer to initiate DNA synthesis at

origins of replication

Primers: Role in Replication

• Primer = short RNA segment complementary to DNA at origins of replication synthesized by primase

• Primer provides free 3’-OH which can be extended by DNA polymerase

• Okazaki fragments are also initiated by primers eventually replaced by DNA; DNA ligase joins ends

DNA Replication: Proteins

• Topoisomerases: nick and unwind DNA to permit strand separation

• RNA primase: initiates strand synthesis by forming RNA primer

• Helicase: unwinds DNA at replication fork • Single-strand binding protein: stabilize

DNA at replication fork

DNA Replication: Proteins

• DNA polymerase complex : catalyzes the incorporation of DNA nucleotides n 3’-to-5’ direction

• DNA ligase: joins Okazaki fragments on lagging strand

Nucleic Acid Hybridization

• DNA denaturation = strand separation occurs by heat to break hydrogen bonds between DNA bases

• DNA renaturation = hybridization = complementary single strands pair and hydrogen bonds form

• Hybridization does not occur unless DNA bases are complementary

Restriction Enzymes

• Restriction enzymes make site specific cleavages in each DNA strand to generate “nicked” single strands with new 5’ and 3’ ends

• Many enzymes cut each DNA strand at different base sites with “staggered cleavages” creating short unpaired “sticky” ends

Southern Blot Analysis• DNA bands on a gel can often be visualized by

staining with dyes which bind DNA (ethidium bromide)

• Southern blot analysis is used to detect very small amounts of DNA or to identify a single DNA band

• Southern blots use labeled “probes” to identify bands by hybridization to complementary DNA bases

Southern Blot Analysis

Steps in Southern blot procedure:• DNA is cut into pieces by restriction

enzymes• DNA fragments are separated by gel

electrophoresis• DNA is transferred from gel to

hybridization filter =blot procedure and denatured to produce single-strand bands of DNA

Southern Blot Analysis

• Filter is mixed with radiolabeled single-stranded DNA probe complementary to the DNA sequence at high temperatures which permit hybridization = hydrogen bonds form between complementary base pairs

• DNA bands hybridized to probe are detected by X-ray film exposure

Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR) is used to detect and amplify very small amounts of DNA

• DNA sequence to be amplified is targeted using

• primer oligonucleotides = short synthetic single-stranded DNA segments complementary to the DNA sequence flanking the region to be amplified

Polymerase Chain Reaction

• Multiple short cycles of replication occurring within the region flanked by primers occurs by controlled temperature shifts which result in repetitive rounds of:

- primer hybridization

- DNA replication of the targeted region by primer extension

- strand separation

Polymerase Chain Reaction

• DNA polymerase used in PCR = Taq polymerase isolated from bacterial thermophiles which can withstand high temperature used in procedure

• PCR accomplishes the rapid production of large amounts of target DNA which can then be identified and analyzed

DNA Sequence Analysis• DNA sequence analysis determines the order

of bases in DNA • Dideoxy method uses DNA bases containing

modified deoxyribose sugars = dideoxyribose which contain H at the 3’ position of the ribose sugar rather than OH

• Modified sugars cause chain termination

Dideoxy Method: DNA Sequencing

• Each of four reactions contains a different dideoxynucleotide = A, T, G, or C in addition to the four bases

• Synthesis occurs in each reaction tube until a dideoxy base is inserted which results in chain termination

• Each tube contains a set of DNA pieces ending with the same base

Dideoxy Method: DNA Sequencing

• Gel electrophoresis is used to separate the reaction products from each tube = DNAs end in A, G, T or C

• DNA sequence can be read in the 5’-to-3’ direction from the bottom of gel

• Each band on the gel is one base longer than the previous band

• Bases are identified by gel position