Ross P M, Yu H S
Rockefeller University, New York, NY 10021.
J Mol Biol. 1988 May 20;201(2):339-51. doi: 10.1016/0022-2836(88)90142-8.
We have used low-level photocrosslinkage to study chromatin effects on psoralen intercalation at specific DNA sequences of various complexities in intact, cultured, Drosophila cells. Alkali-denatured DNA connected in both strands to a 4,5',8-trimethylpsoralen (TMP) interstrand crosslink is insensitive to digestion by the single strand-specific nuclease S1 and does not hybridize to complementary DNA. Crosslink number at any ultraviolet light exposure increases in proportion to the concentration [PS] of TMP dark binding sites that are occupied. The crosslinking constant, K, is the increase in crosslink number per length DNA per increment [PS]. Many factors influence K, including sequence composition and ionic strength. We show here that the ratio of K at any specific sequence (Kh, from hybridization measurements) to Kh at any other specific sequence or to K of total DNA (Kf, from fluorimetry measurements) can be calculated from measurements of crosslinkage, the mass fraction of the sequence in question or of total DNA that is connected in both strands to a crosslink. When crosslinked and uncrosslinked DNAs fragmented by mechanical shear were mixed in known proportions, Kf exceeded Kh of a single-copy gene by 15%. We treated cells with TMP plus near ultraviolet light, then tested for crosslinkage and for hybridization. A single-copy, larval gene at 70D, and a 250-copy type 1 ribosomal DNA intervening sequence, neither of which is transcribed in these cells, were as sensitive to crosslinkage as total, cell DNA. However, single-copy, heat shock gene sequences from loci 63BC and 95D, and the 180-copy ribosomal DNA coding sequence were more sensitive to crosslinkage than total DNA in the same preparations. The excess was largest in the shortest fragments, indicating a localized effect. The same sequences were crosslinked less readily than total DNA in vitro; we calculate a 3.4 to 3.8-fold excess crosslink number in these sequences due to chromatin microenvironment. We tested for effect of transcriptional induction on crosslink sensitivity in the heat shock genes. At low [TMP], heat shock stimulated crosslinkage at or very near heat shock genes in cells, but not in other sequences or in naked DNA. However, overall crosslink sensitivity was unaffected by heat shock. This suggests that transcription increased the affinity of some heat shock gene DNA binding sites for TMP without increasing the number of such sites.
我们利用低水平光交联来研究染色质对完整的、培养的果蝇细胞中各种复杂程度的特定DNA序列上补骨脂素嵌入的影响。在两条链上都与4,5',8-三甲基补骨脂素（TMP）链间交联相连的碱变性DNA对单链特异性核酸酶S1的消化不敏感，并且不与互补DNA杂交。在任何紫外线照射下，交联数与被占据的TMP暗结合位点的浓度[PS]成比例增加。交联常数K是每增加[PS]时每长度DNA交联数的增加量。许多因素会影响K，包括序列组成和离子强度。我们在此表明，任何特定序列处的K（通过杂交测量得到的Kh）与任何其他特定序列处的Kh或总DNA的K（通过荧光测定得到的Kf）的比值，可以通过交联测量、所研究序列或与交联在两条链上相连的总DNA的质量分数来计算。当通过机械剪切破碎的交联和未交联DNA以已知比例混合时，Kf比单拷贝基因的Kh高15%。我们用TMP加近紫外光处理细胞，然后检测交联和杂交情况。位于70D的单拷贝幼虫基因和一个250拷贝的1型核糖体DNA间隔序列，在这些细胞中均不转录，它们对交联的敏感性与细胞总DNA相同。然而，来自63BC和95D位点的单拷贝热休克基因序列以及180拷贝的核糖体DNA编码序列在相同制剂中对交联的敏感性比总DNA更高。这种过量在最短的片段中最大，表明存在局部效应。相同的序列在体外比总DNA更难交联；由于染色质微环境，我们计算出这些序列中的交联数过量3.4至3.8倍。我们测试了转录诱导对热休克基因交联敏感性的影响。在低[TMP]时，热休克刺激了细胞中热休克基因处或非常接近热休克基因处的交联，但在其他序列或裸DNA中没有。然而，总体交联敏感性不受热休克影响。这表明转录增加了一些热休克基因DNA结合位点对TMP的亲和力，但没有增加此类位点的数量。
