? ? 本期的內(nèi)容是第五章：DNA復(fù)制的前兩節(jié)內(nèi)容：實(shí)驗(yàn)、復(fù)制過(guò)程。如果有不太明白的或者有錯(cuò)誤的地方隨時(shí)來(lái)找UP主喔~ 文集本部分的參考文獻(xiàn)Essential Cell Biology, 5th ed. Alberts, et al. 2019. 部分內(nèi)容來(lái)自khanacademy與維基百科.

? ? 本期封面/頭圖：https://www.bing.com/images/search?view=detailV2&ccid=13TyVcCB&id=0DC14A19ADBF0D18D83EFCC96D336607669A1883&thid=OIP.13TyVcCBsJ91st7lohMMqAHaEK&mediaurl=https%3A%2F%2Fi.ytimg.com%2Fvi%2FcwRzplFt6A8%2Fmaxresdefault.jpg&exph=720&expw=1280&q=DNA+Replication&simid=608036794161496114&ck=EFE3FEF7EF06C6B1B8FAC23BE9061DDD&selectedindex=6&form=IRPRST&ajaxhist=0&vt=0&sim=11

本期內(nèi)容的筆記以文本的形式呈現(xiàn)。

L5 Replication

5a Replication: Key Experiments

????DNA Replication: complementary pairing DNA structure told people something about the replication, but not sufficient

3 models for replication:

????Semiconservative model: old strands separate, copy & stay with the new strand

????Dispersive model: each strand is composed by short segments of old & new

????Conservative model: original strands copy & reassemble, they are conserved

Meselson and Stahl, 1958

????To distinguish old DNA from new

????Grow bacteria in media (liquid food) with heavy (bacteria grown in 15N?(氮15)-containing medium) or light N (bacteria grown in 14N?(氮14)-containing medium)

????Heavy: bacteria grown incorporate 15N (氮15)?in the DNA

????Light:bacteria grown incorporate 14N?(氮14) in the DNA

How to distinguish molecules that have different weights and densities?

????- Density centralization: Put N-DNA into the tubes, then spin.

Experimental Procedures

????1) Grow cells for many generations in the heavy medium (15N)

????2) Transfer cells to light medium (14N)

????3) Grow cells for one or two more replication cycles

????4) Isolate DNA

Different Models should give different results -?Prediction of the semiconservative model:

This pattern was seen in experiments:?third?generation: top line 75%, middle line 25%

5b Replication: Copying the strands

Replication - Essential Components

????1. Template DNA: DNA that wants to copy

? ? 2. dNTPs: deoxynucleoside triphosphate

????????- As subunits?- covalently linking together as a polymer

????????- As energy?- source of energy for the replication reaction

? ? 3. DNA Polymerase - enzyme that catalyzes the synthesis the DNA

? ??4. Other proteins

Initiation of DNA replication

????- Requires unwinding the double helix?

????- Start at the Replication Origin (ori) - then local opening of DNA helix?

????- The replication origin is A-T rich. Because the A-T pairs have fewer H-bonds, easier to separate the strands.

As the replication bubble gets bigger, two replication forks move away from each other; thus, more and more double-stranded DNA unzips.

Eukarytic Chromosome - many origins Replication bubbles will meet together

Elongation

????A dNTP base-pairs to the template

????????- Note: NTP = any Nucleotide Tri-Phosphate,?dNTP= the deoxy version (DNA, not RNA)

????DNA Polymerase synthesizes by attaching dNTPs onto 3’-OH groups?

????????- Synthesis is 5’ to 3’; the template is read 3’ to 5’.?

????????- High energy bond break (downhill), pyrophosphate (PPi) is released.

DNA synthesis requires energy

????- Condensation reaction?

????- Hydrolyzing nucleotide tri-phosphate

Energy source for replication:

????- Deoxynucleotides (ATP is an RNA nucleotide)

????- Any base, not just Adenine

????????Also dATP, dCTP, dGTP, dTTP

????- Nucleotide cleaved between 1st and 2nd phosphates

????????ATP is cleaved between 2nd and 3rd phosphates

DNA polymerase needs a 3’OH to be able to add nucleotides.

????RNA Primer - complement to the DNA template to start

dAMP is missing both the 2nd and 3rd phosphate group on the 5' carbon. Normally, the energy released from cleavage of the bonds between phosphates is what allows the addition of a nucleotide to the growing chain. If you can't get energy for DNA synthesis, this uphill condensation reaction cannot occur, and the dAMP nucleotide will not be attached.

ddATP (dideoxy ATP) has 3 phosphates at its 5' end, so one ddATP can be covalently attached to the growing strand. However, ddATP is missing its 3' OH. The DNA polymerase needs the nucleotide at the end of the growing DNA strand to have a 3' OH on which to attach the next nucleotide. Therefore one ddATP will be attached to the growing strand, but replication will cease after that. ddNTPs are called "chain terminators."

Replication bubble

????DNA on the leading strand is synthesized from 5’ to 3’, same direction as the fork movement.

????DNA on the lagging strand is synthesized in the opposite direction as the fork movement and in discontinuous fragments (Okazaki fragments), from 5’ to 3’.

????Lagging strand polymerase repeatedly returns to the fork, and starts synthesizing away from it again.

????Rules of replication: 1) always 5’ to 3’, template read 3’ to 5’; 2) start with an RNA primer; 3) two replication forks, four places that replication happen.

本次內(nèi)容到此結(jié)束，感謝閱讀！之后的內(nèi)容將講述DNA復(fù)制過(guò)程中的蛋白質(zhì)與其他活動(dòng)。

作者：離久-張所長(zhǎng)